Overview of the flow cytometer. 2003, 23 (7): 369-377. Comparative results are shown with backgrounds subtracted; no significant differences in backgrounds were seen with liquid versus lyophilised reagents (data not shown). Edited by: Hawley TS, Hawley RG. De Rosa SC, Lu FX, Yu J, Perfetto SP, Falloon J, Moser S, Evans TG, Koup R, Miller CJ, Roederer M: Vaccination in humans generates broad T cell cytokine responses. It allows simultaneous multi-parameter analysis of single cells. Viral Immunol. versus mean for all assays. Like the fresh whole blood experiment, the improvement in C.V. from centralized analysis was relatively small. CEF was expected to induce CD8, rather than CD4 responses, and indeed the CD4 responses to this control were very low or negative. After 10 minutes at room temperature, the tubes were frozen at -80C, then shipped to participating laboratories on dry ice. (A) Data from one site that compared cocktail 1 (CD4 FITC/IFN +IL-2 PE/CD8 PerCP-Cy5.5/CD3 APC) in liquid and lyophilised form in Experiment 4. 2000, 7 (3): 344-351. Holden T Maecker. Article 
For a positive result you are looking for the shift in intensity between negative control and a positive samples (Figure 9). Cryopreserved PBMC are much more amenable to peptide (and superantigen) stimulation than to whole protein stimulation [9]; while whole blood assays are equally amenable to stimulation with either type of antigen. Raw data were also sent to a central site for batch analysis with a dynamic gating template. These cells served as a control for instrument setup and gating, since all the activation and processing steps were done centrally. A useful example of this is when running blood samples on the flow cytometer. This could be due to the inclusion of IL-2 in cocktail 1, which would be expected to be produced by more CD4+ than CD8+ cells, and thus contribute selectively to the CD4 background. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 520%, depending upon the experiment. In a second experiment, whole blood from three CMV-seropositive donors was shipped overnight to 6 U.S. labs for activation, processing, and analysis. P.D'S. This conclusion cannot be drawn with certainty, because different participating laboratories and different donors were used between experiments 3 and 4. It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA. Figure 2. Data is from an anonymous Abreview. However, when the raw data was centrally analyzed, the inter-lab C.V. was reduced to 24% for both CD4 and CD8 T cell responses, very similar to the inter-assay C.V. previously reported [6]. The relative fluorochrome intensity depends on the instrument. Comparison of liquid and lyophilised reagents. Forward and side scattered light is detected, as well as fluorescence emitted from stained cells. All in all, the choice of assay format for a clinical trial will depend not only upon considerations of assay precision, but also upon the type of antigen(s) used and the capabilities of the participating clinical sites. Various filters are used in the flow cytometer to direct photons of the correct wavelength to each PMT (Figure 5). Backgrounds were generally low. Cells or particles passing through the beam scatter light, which is detected as FS and SS. The improvement in C.V. made by centralized analysis was most marked in the first experiment, and progressively less in experiments 2 and 3, perhaps because of standardization of gating among sites over time. The difference in average C.V. between cocktail 1 and cocktails 2 and 3 was preserved even after centralized analysis. Whole blood was activated with either SEB or pp65 peptide mix, and the percentage of IFN+ cells (CD4+ or CD8+) were compared with liquid versus lyophilised reagents (left panel). Manage cookies/Do not sell my data we use in the preference centre. Responses to CMV pp65 peptide mix were also not high in the donors used in this experiment, but were detectable in both CD4 and CD8 compartments. California Privacy Statement, Clin Diagn Lab Immunol.
Intracellular Staining for Flow Cytometry How-To Videofor detecting cytokines and intranuclear markers. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Holden T Maecker,Maria A Suni&Vernon C Maino, Universit de Montreal and CANVAC, the Canadian Network for Vaccines and Immunotherapeutics, Montreal, Canada, Aline Rinfret,Eva Roig,Claire Landry&Rafick P Sekaly, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA, Chelsea and Westminster Hospital and IAVI, London, UK, Uganda Virus Research Institute and IAVI, Entebbe, Uganda, Kenya AIDS Vaccine Initiative (KAVI), University of Nairobi, Kenya, Centre Hospitalier Universitaire Vaudois and EUROVAC, Lausanne, Switzerland, Miguel Garcia,Alexandre Harari&Giuseppe Pantaleo, University of Washington and HVTN, Fred Hutchinson Cancer Research Center, Seattle, USA, Duke University Medical Center and HVTN, Durham, USA, Megan Baker,Jennifer Holbrook,Janet Ottinger&Kent Weinhold, Vaccine Research Center, National Institutes of Health, Bethesda, USA, Laurie Lamoreaux,Mario Roederer&Richard Koup, University of California, San Francisco, USA, C Lorrie Epling,Elizabeth Sinclair&Barry Bredt, University of Pennsylvania, Philadelphia, USA, Massachusetts General Hospital, Boston, USA, National Institute for Communicable Diseases, Johannesburg, South Africa, You can also search for this author in PubMedGoogle Scholar. By using this website, you agree to our Figure 3. Fluorescent detection of -galactosidase hydrolysis by flow cytometry. In the example shown in Figure 4, the FITC (fluorescein isothiocyanate) channel PMT will detect light emitted from FITC at a wavelength of approximately 519 nm.
Create Account, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Microbiological Media and Media Additives, Gel Electrophoresis Equipment and Supplies, Immunophenotyping Kits for Flow Cytometry, Flow Cytometry Beads for Instrument Controls, Standards, and Compensation, Flow Cytometry Sample Preparation Buffers and Reagents, Spectral Flow Cytometry Assays and Reagents, Brilliant Ultra Violet and Brilliant Violet Polymer Dyes, Ask our technical specialists which you should purchase and how to incorporate into your experiment, Learn more about the Invitrogen Attune NxT flow cytometer here, Dye dilution assays for cell proliferation, Live cell oxidative stress assays for flow cytometry, Plasma membrane assays, annexin V staining, Immunophenotyping kits for flow cytometry, Beads for cell counting, instrument set-up, and compensation, Intracellular Staining for Flow Cytometry How-To Video, 5 Steps to Publication-Quality Fixed Cell Imaging. J Assoc Lab Automation. The median CD8 background in the PBMC experiments was significantly lower than that of the frozen activated whole blood experiment (p < 0.0001) or the fresh whole blood experiment (p < 0.05). These data should guide comparisons of ICS results between different groups or in different clinical trials. For comparison, the inter-lab C.V. of the CD4+ or CD8+ cell percentages was around 5% in experiment 4 of the present study (data not shown). For experiment 2, 5 mL of heparinized whole blood was overnight shipped in an insulated container at ambient temperature to each participating lab. Simultaneous detection of RNA and proteins by flow cytometry using the PrimeFlow RNA Assay. It was later determined that cross-contamination probably occurred during the initial distribution of the antigens on the plates, and this was compounded by the fact that the donors used were unusually sensitive to SEB stimulation (responses >30% of CD4+ and CD8+ T cells). Article The inter-lab C.V. was lowest (1824%) for samples with a mean of >0.5% IFN + T cells, and highest (5782%) for samples with a mean of <0.1% IFN + cells. Results of Experiment 2 (shipped whole blood). AR coordinated the study. The average C.V. across the four experiments is summarized in Table 3. Nat Med. Article Figure 5. Google Scholar. In addition, a protocol refinement was introduced to attempt to further reduce inter-lab variability: Peptide stimuli together with brefeldin A were provided in lyophilised form in appropriate wells of a microtiter plate, to provide simplified assay set-up; and lyophilised staining antibody cocktails were provided in the corresponding wells of a second microtiter plate. Not for use in diagnostic procedures. ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. For example, (Z +Z)2 = 23.9 for 99% power and p < 0.005 [23]. Be sure to use the appropriate FACS instrument. Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations, and analyzing cell size and volume. Results from each site are indicated as a circle, with median responses for each sample indicated by a horizontal bar. Original FCS files from each site were sent to BD Biosciences for analysis using a dynamic gating template (Figure 2B). An antigen expressed at lower density might require the higher signal to background ratio provided by a brighter PE or APC conjugate to separate the positive cells adequately from the unstained cells. These data are confounded by the fact that different donors and different laboratories participated in the four experiments. The channels are usually viewed on a log scale on the x axis. In addition to convenience of assay set-up, the lyophilised reagent plates offer long reagent stability, even at room temperature (>1 year, data not shown), and a potential reduction in errors caused by incorrect reagent addition. We offer a series of fluorescent reagents designed for accurate cell cycle analysis in either live- or fixed-cell populations. As each fluorescing cell completes its path through the laser beam, this leaves a pulse of voltage over time. Pantaleo G, Koup RA: Correlates of immune protection in HIV-1 infection: what we know, what we don't know, what we should know. ICS is also comparable to cytokine ELISA, the latter having reported interassay C.V.'s of <25% [31, 32]. Mitigation of gating variability was achieved in these experiments by centralized analysis with a dynamic gating template (see Figure 2B).
When no fluorescing cells pass through the optics, no photons are emitted and no signal is detected. J Interferon Cytokine Res. Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, Group ELISPOTS: Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. These cryopreserved PBMC were shipped to participating labs using liquid nitrogen dry shippers. Mean viabilities were >82%, and mean recoveries were >75% for each sample (determined by trypan blue exclusion). Take a look at our BETA site and see what weve done so far. Intra-plate variability using lyophilised reagents was determined to be <10% in ICS assays (data not shown). 2003, Sept.: 34-41. Maecker, H.T., Rinfret, A., D'Souza, P. et al. Findlay JW, Smith WC, Lee JW, Nordblom GD, Das I, DeSilva BS, Khan MN, Bowsher RR: Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective. Sample names are listed along the X axis. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays. However, this could be due to the fact that different donors were used in the four experiments, rather than being due to any inherent difference between assay types. 
These reagents and assays can be incorporated into a flow cytometry panel just like a flow cytometry antibody. Sun Y, Iglesias E, Samri A, Kamkamidze G, Decoville T, Carcelain G, Autran B: A systematic comparison of methods to measure HIV-1 specific CD8 T cells. Cytometry. In some labs, automated setup was followed by manual adjustment using stained cells as above. Thermo Fisher Scientific. The results, as reported by each site and also as determined by central, automated analysis of the raw data files, are summarized in Figure 3. Figure 4. Numerous phase I and phase II clinical trials have been initiated using candidate prophylactic HIV vaccines (reviewed in [4]). In this case, a fluorochrome is often used to stain the protein of interest. Aids. These fluorescent stained cells or particles can be detected individually. Biopharm Intl.
The results reported by the individual sites for these cells are shown in the left panel of Figure 7C. Moretto WJ, Drohan LA, Nixon DF: Rapid quantification of SIV-specific CD8 T cell responses with recombinant vaccinia virus ELISPOT or cytokine flow cytometry.
Fluorochromes used for the detection of target proteins emit light when excited by a laser with the corresponding excitation wavelength. Cell death cascades are complex and dynamic, underscoring the importance of a multi-parametric approach to apoptosis detection. These have been extensively compared to liquid reagents ([22] and Figure 9) and shown to be largely equivalent. Because many different groups are performing immune monitoring for these clinical trials, there is currently a lack of standardization that would allow accurate comparisons of immunogenicity across candidate vaccines in different clinical trials. https://doi.org/10.1186/1471-2172-6-13, DOI: https://doi.org/10.1186/1471-2172-6-13. For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome. Larger and more granular granulocyte cells produce a large population with high SS and FS. Cite this article. The C.V. increased further at response levels of =0.1%. However, improved precision of ICS results might be obtained if certain acceptance criteria were applied before data were taken as valid. Smaller lymphocytes and lymphoblasts produce a separate population with less FS. There are some potential drawbacks to the use of 96-well plates.
IFN-positive cells in response to SEB or CMV pp65 peptide mix are expressed as a percentage of CD4+ or CD8+ T cells. This could reflect the fact that new laboratory sites had been added that had not yet standardized their gating strategies with the existing sites; thus more benefit was realized by centralized analysis. Search Despite the lower response means, the average C.V. for this experiment was roughly similar to the previous experiment, when comparing the same staining antibody cocktails (cocktails 2 and 3). For experiment 1, the blood was activated in 15 mL conical tubes according to the method of Nomura et al.[6]. Weinberg A, Zhang L, Brown D, Erice A, Polsky B, Hirsch MS, Owens S, Lamb K: Viability and functional activity of cryopreserved mononuclear cells. Cocktail 1 consisted of CD4 FITC/IFN +IL-2 PE/CD8 PerCP-Cy5.5/CD3 APC. CD4 counting precision has also been shown to be dependent upon the number of events collected, gating, and use of automated analysis [33, 34]. Standardization of cytokine flow cytometry assays. Maecker HT, Dunn HS, Suni MA, Khatamzas E, Pitcher CJ, Bunde T, Persaud N, Trigona W, Fu TM, Sinclair E, Bredt BM, McCune JM, Maino VC, Kern F, Picker LJ: Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. These are detected by the PMT and converted to a voltage pulse, known as an event.
2003, 283 (1-2): 141-153. If you arelooking to get to grips with flow cytometry analysis, check out our free online flow cytometry training. A general guideline for the relative intensities of the various fluorochromes is, from brightest to dimmest, PE, PE-Cy 7, PE-Cy5, APC, APC-Cy7, Alexa Fluor 647, Alexa Fluor 700, FITC, Pacific Blue, Alexa Fluor 488. A set of peptides consisting of epitopes from CMV, EBV, and influenza (CEF) [13] was used as a positive control, due to restrictions on international shipment of SEB. A significant portion of inter-laboratory variability could be eliminated by use of centralized analysis and/or a dynamic gating template. In order to allow more meaningful comparisons between laboratories and prioritization of emerging vaccine candidates, and thereby accelerate HIV vaccine development, this ICS standardization study was undertaken. With the possibility of achieving inter-laboratory C.V.'s of less than 20%, even with relatively low responses, ICS compares favourably to ELISPOT, for which interassay C.V.'s of 1718% for PHA and 5565% for Candida have been reported [29, 30]. However, variability due to individual analysis can be excluded by comparing only centrally analysed data (bottom row of Table 3). The PE channel PMT will detect light emitted from PE (phycoerythrin) at 575 nm wavelength. Sample names are listed along the X axis. While CD4 backgrounds were very similar between experiments, CD8 backgrounds varied. Instrument setup was at the discretion of the individual laboratory, and was either manual (using isotype control stained cells to set PMT voltages, and single-stained cells to set compensation) or automated (using BD FACSComp software and BD Calibrite beads (BD Biosciences)). When centralized automated analysis was applied to the data, backgrounds were not usually reduced. Some of these sites experienced high backgrounds in the negative control wells adjacent to the SEB-containing wells. Cocktail 2 consisted of IFN FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC, and cocktail 3 consisted of IFN FITC/CD69 PE/CD8 PerCP-Cy5.5/CD3 APC. Note that the data points for all three assay types cluster together, indicating that variability is similar for all three assay types. 2004, Wayne, PA , NCCLS, I/LA26-A. The inter-lab C.V.'s for this experiment averaged slightly lower than those for the whole blood experiments (2532% for manual analysis; 2325% for centralized automated analysis). Manual versus automated gating templates. T cell production of IFN, and increasingly also IL-2, is taken as a measure of vaccine immunogenicity in experimental vaccine trials. statement and Forward and side scattered light and fluorescence from stained cells are split into defined wavelengths and channeled by a set of filters and mirrors within the flow cytometer. Flow Cytometry Support CenterFind technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help. With the high viabilities and recoveries obtained for cryopreserved PBMC in this study, there was no obvious relationship between viability/recovery and response. Clin Chem Lab Med. The percent CD69+IFNg+ cells obtained is very similar to that obtained by manual gating in this example, since manual gating was performed so as to include CD3dim and CD8dim cells. Once such a template was created and optimized (see Materials and Methods section for description), it could also have been provided to individual sites in order to yield the same results. 2000, 25 (2): 109-114. There were a significant number of CD8 samples that exhibited high spontaneous cytokine production. Kleeberger CA, Lyles RH, Margolick JB, Rinaldo CR, Phair JP, Giorgi JV: Viability and recovery of peripheral blood mononuclear cells cryopreserved for up to 12 years in a multicenter study.
The use of microtiter plates containing lyophilised reagents simplified the ICS protocol, and appeared to improve assay reproducibility. The results of the second trial are shown in Figure 4. PubMed 2000, 14 (16): 2625-2627. In a long pass dichroic filter, photons above a specific wavelength are transmitted straight ahead, whilst photons below the specific wavelength are reflected at a 90 angle. Blood. The template was then used, without further adjustment, on all the files of a given experiment. The PMTs convert the energy of a photon into an electronic signal a voltage. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Results from each site are indicated as a circle, with median responses for each sample indicated as a horizontal bar.
(C) Control cell data from Experiment 4. 10 million per second or 10 per microsecond). Clin Diagn Lab Immunol. In fact, there was no obvious relationship of viability or recovery with response, perhaps because viabilities and recoveries were virtually all within generally acknowledged limits of acceptability (>80% viability, >50% recovery) [11, 12]. Sheath fluid focuses the cell suspension, causing cells to pass through a laser beam one cell at a time. For example, a minimum number of collected events could be specified (sites in this study were asked to collect 10,00040,000 CD4+ or CD8+ T cells per sample, or 60,000 CD3+ cells). Figure 1. When SEB was replaced with CEF as a positive control, no such problems were noted. Flow Cytometry Learning CenterAccess flow cytometry educational resources for better experiment planning and execution. Provided by the Springer Nature SharedIt content-sharing initiative. The blood was incubated for 6 hours in the presence of brefeldin A, either with no stimulus, Staphylococcal enterotoxin B (SEB), or a mixture of overlapping peptides corresponding to the CMV pp65 protein [810].
The Invitrogen Immunology at Work Resource Center is a learning center with technical content designed for new and experienced life scientists alike exploring the field of immunology. Results from each site are indicated as a circle, with median responses for each sample (105, 1040, and 1090) indicated by a horizontal bar. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Data from experiment 4 are for cocktails 2 and 3 only, to be comparable with experiments 13. This format lends itself to international shipping of reagents (because there is no need for refrigeration), and also to larger clinical trials (because of the stability of the lyophilised reagents). 2003, March: 42-50. Trying to incorporate a reagent into your panel?Ask our technical specialists which you should purchase and how to incorporate into your experiment, Need to add a flow cytometer to your lab?Learn more about the Invitrogen Attune NxT flow cytometer here. This experiment also sought to compare two different types of staining antibody cocktails: the two cocktails used in the previous three experiments (IFN FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC and IFN FITC/CD69 PE/CD8 PerCP-Cy5.5/CD3); and one that combined CD4 and CD8 staining in a single sample, as well as adding IL-2 staining (CD4 FITC/IFN +IL-2 PE/CD8 PerCP-Cy5.5/CD3 APC). (A) Data reported by individual sites. The C.V. for each sample is listed across the top of each panel, along with the mean C.V. for that set of samples. As a positive control in Experiment 4, a set of PBMC were SEB-activated, processed, stained, and then lyophilised in certain wells of the lyophilised antibody plates. These values were determined to be about 8% and 20% C.V., respectively. For Research Use Only. Viabilities and recoveries for cryopreserved PBMC samples used in Experiment 3. The CD8 responses, while positive, were considerably lower than those seen with SEB in the previous experiments.
Replicate cryopreserved vials were sent to each of 7 sites, where they were thawed, rested overnight, stimulated, processed, and analyzed. This is a general pattern; some differences in the relative intensities are seen for individual antibodies. All rights reserved. J Immunol Methods. (A) Schematic of protocol for Experiments 13, performed using liquid antigens and antibodies. Figure 8. 2002, 185 (12): 1709-1716. The post-thaw viability and recovery of the PBMC samples from each site are shown in Figure 5. Dot plot of FS versus SS. These sensors are called photomultiplying tubes (PMTs). Cytokine-positive cells in the absence of stimulus are expressed as a percentage of CD4+ or CD8+ T cells.
For a positive result you are looking for the shift in intensity between negative control and a positive samples (Figure 9). Cryopreserved PBMC are much more amenable to peptide (and superantigen) stimulation than to whole protein stimulation [9]; while whole blood assays are equally amenable to stimulation with either type of antigen. Raw data were also sent to a central site for batch analysis with a dynamic gating template. These cells served as a control for instrument setup and gating, since all the activation and processing steps were done centrally. A useful example of this is when running blood samples on the flow cytometer. This could be due to the inclusion of IL-2 in cocktail 1, which would be expected to be produced by more CD4+ than CD8+ cells, and thus contribute selectively to the CD4 background. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 520%, depending upon the experiment. In a second experiment, whole blood from three CMV-seropositive donors was shipped overnight to 6 U.S. labs for activation, processing, and analysis. P.D'S. This conclusion cannot be drawn with certainty, because different participating laboratories and different donors were used between experiments 3 and 4. It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA. Figure 2. Data is from an anonymous Abreview. However, when the raw data was centrally analyzed, the inter-lab C.V. was reduced to 24% for both CD4 and CD8 T cell responses, very similar to the inter-assay C.V. previously reported [6]. The relative fluorochrome intensity depends on the instrument. Comparison of liquid and lyophilised reagents. Forward and side scattered light is detected, as well as fluorescence emitted from stained cells. All in all, the choice of assay format for a clinical trial will depend not only upon considerations of assay precision, but also upon the type of antigen(s) used and the capabilities of the participating clinical sites. Various filters are used in the flow cytometer to direct photons of the correct wavelength to each PMT (Figure 5). Backgrounds were generally low. Cells or particles passing through the beam scatter light, which is detected as FS and SS. The improvement in C.V. made by centralized analysis was most marked in the first experiment, and progressively less in experiments 2 and 3, perhaps because of standardization of gating among sites over time. The difference in average C.V. between cocktail 1 and cocktails 2 and 3 was preserved even after centralized analysis. Whole blood was activated with either SEB or pp65 peptide mix, and the percentage of IFN+ cells (CD4+ or CD8+) were compared with liquid versus lyophilised reagents (left panel). Manage cookies/Do not sell my data we use in the preference centre. Responses to CMV pp65 peptide mix were also not high in the donors used in this experiment, but were detectable in both CD4 and CD8 compartments. California Privacy Statement, Clin Diagn Lab Immunol. Intracellular Staining for Flow Cytometry How-To Videofor detecting cytokines and intranuclear markers. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Holden T Maecker,Maria A Suni&Vernon C Maino, Universit de Montreal and CANVAC, the Canadian Network for Vaccines and Immunotherapeutics, Montreal, Canada, Aline Rinfret,Eva Roig,Claire Landry&Rafick P Sekaly, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA, Chelsea and Westminster Hospital and IAVI, London, UK, Uganda Virus Research Institute and IAVI, Entebbe, Uganda, Kenya AIDS Vaccine Initiative (KAVI), University of Nairobi, Kenya, Centre Hospitalier Universitaire Vaudois and EUROVAC, Lausanne, Switzerland, Miguel Garcia,Alexandre Harari&Giuseppe Pantaleo, University of Washington and HVTN, Fred Hutchinson Cancer Research Center, Seattle, USA, Duke University Medical Center and HVTN, Durham, USA, Megan Baker,Jennifer Holbrook,Janet Ottinger&Kent Weinhold, Vaccine Research Center, National Institutes of Health, Bethesda, USA, Laurie Lamoreaux,Mario Roederer&Richard Koup, University of California, San Francisco, USA, C Lorrie Epling,Elizabeth Sinclair&Barry Bredt, University of Pennsylvania, Philadelphia, USA, Massachusetts General Hospital, Boston, USA, National Institute for Communicable Diseases, Johannesburg, South Africa, You can also search for this author in PubMedGoogle Scholar. By using this website, you agree to our Figure 3. Fluorescent detection of -galactosidase hydrolysis by flow cytometry. In the example shown in Figure 4, the FITC (fluorescein isothiocyanate) channel PMT will detect light emitted from FITC at a wavelength of approximately 519 nm.
Create Account, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Microbiological Media and Media Additives, Gel Electrophoresis Equipment and Supplies, Immunophenotyping Kits for Flow Cytometry, Flow Cytometry Beads for Instrument Controls, Standards, and Compensation, Flow Cytometry Sample Preparation Buffers and Reagents, Spectral Flow Cytometry Assays and Reagents, Brilliant Ultra Violet and Brilliant Violet Polymer Dyes, Ask our technical specialists which you should purchase and how to incorporate into your experiment, Learn more about the Invitrogen Attune NxT flow cytometer here, Dye dilution assays for cell proliferation, Live cell oxidative stress assays for flow cytometry, Plasma membrane assays, annexin V staining, Immunophenotyping kits for flow cytometry, Beads for cell counting, instrument set-up, and compensation, Intracellular Staining for Flow Cytometry How-To Video, 5 Steps to Publication-Quality Fixed Cell Imaging. J Assoc Lab Automation. The median CD8 background in the PBMC experiments was significantly lower than that of the frozen activated whole blood experiment (p < 0.0001) or the fresh whole blood experiment (p < 0.05). These data should guide comparisons of ICS results between different groups or in different clinical trials. For comparison, the inter-lab C.V. of the CD4+ or CD8+ cell percentages was around 5% in experiment 4 of the present study (data not shown). For experiment 2, 5 mL of heparinized whole blood was overnight shipped in an insulated container at ambient temperature to each participating lab. Simultaneous detection of RNA and proteins by flow cytometry using the PrimeFlow RNA Assay. It was later determined that cross-contamination probably occurred during the initial distribution of the antigens on the plates, and this was compounded by the fact that the donors used were unusually sensitive to SEB stimulation (responses >30% of CD4+ and CD8+ T cells). Article The inter-lab C.V. was lowest (1824%) for samples with a mean of >0.5% IFN + T cells, and highest (5782%) for samples with a mean of <0.1% IFN + cells. Results of Experiment 2 (shipped whole blood). AR coordinated the study. The average C.V. across the four experiments is summarized in Table 3. Nat Med. Article Figure 5. Google Scholar. In addition, a protocol refinement was introduced to attempt to further reduce inter-lab variability: Peptide stimuli together with brefeldin A were provided in lyophilised form in appropriate wells of a microtiter plate, to provide simplified assay set-up; and lyophilised staining antibody cocktails were provided in the corresponding wells of a second microtiter plate. Not for use in diagnostic procedures. ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. For example, (Z +Z)2 = 23.9 for 99% power and p < 0.005 [23]. Be sure to use the appropriate FACS instrument. Flow cytometry is a widely used method for analyzing the expression of cell surface and intracellular molecules, characterizing and defining different cell types in a heterogeneous cell population, assessing the purity of isolated subpopulations, and analyzing cell size and volume. Results from each site are indicated as a circle, with median responses for each sample indicated by a horizontal bar. Original FCS files from each site were sent to BD Biosciences for analysis using a dynamic gating template (Figure 2B). An antigen expressed at lower density might require the higher signal to background ratio provided by a brighter PE or APC conjugate to separate the positive cells adequately from the unstained cells. These data are confounded by the fact that different donors and different laboratories participated in the four experiments. The channels are usually viewed on a log scale on the x axis. In addition to convenience of assay set-up, the lyophilised reagent plates offer long reagent stability, even at room temperature (>1 year, data not shown), and a potential reduction in errors caused by incorrect reagent addition. We offer a series of fluorescent reagents designed for accurate cell cycle analysis in either live- or fixed-cell populations. As each fluorescing cell completes its path through the laser beam, this leaves a pulse of voltage over time. Pantaleo G, Koup RA: Correlates of immune protection in HIV-1 infection: what we know, what we don't know, what we should know. ICS is also comparable to cytokine ELISA, the latter having reported interassay C.V.'s of <25% [31, 32]. Mitigation of gating variability was achieved in these experiments by centralized analysis with a dynamic gating template (see Figure 2B).
When no fluorescing cells pass through the optics, no photons are emitted and no signal is detected. J Interferon Cytokine Res. Cox JH, Ferrari G, Kalams SA, Lopaczynski W, Oden N, Group ELISPOTS: Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. These cryopreserved PBMC were shipped to participating labs using liquid nitrogen dry shippers. Mean viabilities were >82%, and mean recoveries were >75% for each sample (determined by trypan blue exclusion). Take a look at our BETA site and see what weve done so far. Intra-plate variability using lyophilised reagents was determined to be <10% in ICS assays (data not shown). 2003, Sept.: 34-41. Maecker, H.T., Rinfret, A., D'Souza, P. et al. Findlay JW, Smith WC, Lee JW, Nordblom GD, Das I, DeSilva BS, Khan MN, Bowsher RR: Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective. Sample names are listed along the X axis. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays. However, this could be due to the fact that different donors were used in the four experiments, rather than being due to any inherent difference between assay types. These reagents and assays can be incorporated into a flow cytometry panel just like a flow cytometry antibody. Sun Y, Iglesias E, Samri A, Kamkamidze G, Decoville T, Carcelain G, Autran B: A systematic comparison of methods to measure HIV-1 specific CD8 T cells. Cytometry. In some labs, automated setup was followed by manual adjustment using stained cells as above. Thermo Fisher Scientific. The results, as reported by each site and also as determined by central, automated analysis of the raw data files, are summarized in Figure 3. Figure 4. Numerous phase I and phase II clinical trials have been initiated using candidate prophylactic HIV vaccines (reviewed in [4]). In this case, a fluorochrome is often used to stain the protein of interest. Aids. These fluorescent stained cells or particles can be detected individually. Biopharm Intl. The results reported by the individual sites for these cells are shown in the left panel of Figure 7C. Moretto WJ, Drohan LA, Nixon DF: Rapid quantification of SIV-specific CD8 T cell responses with recombinant vaccinia virus ELISPOT or cytokine flow cytometry.
Fluorochromes used for the detection of target proteins emit light when excited by a laser with the corresponding excitation wavelength. Cell death cascades are complex and dynamic, underscoring the importance of a multi-parametric approach to apoptosis detection. These have been extensively compared to liquid reagents ([22] and Figure 9) and shown to be largely equivalent. Because many different groups are performing immune monitoring for these clinical trials, there is currently a lack of standardization that would allow accurate comparisons of immunogenicity across candidate vaccines in different clinical trials. https://doi.org/10.1186/1471-2172-6-13, DOI: https://doi.org/10.1186/1471-2172-6-13. For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome. Larger and more granular granulocyte cells produce a large population with high SS and FS. Cite this article. The C.V. increased further at response levels of =0.1%. However, improved precision of ICS results might be obtained if certain acceptance criteria were applied before data were taken as valid. Smaller lymphocytes and lymphoblasts produce a separate population with less FS. There are some potential drawbacks to the use of 96-well plates.
IFN-positive cells in response to SEB or CMV pp65 peptide mix are expressed as a percentage of CD4+ or CD8+ T cells. This could reflect the fact that new laboratory sites had been added that had not yet standardized their gating strategies with the existing sites; thus more benefit was realized by centralized analysis. Search Despite the lower response means, the average C.V. for this experiment was roughly similar to the previous experiment, when comparing the same staining antibody cocktails (cocktails 2 and 3). For experiment 1, the blood was activated in 15 mL conical tubes according to the method of Nomura et al.[6]. Weinberg A, Zhang L, Brown D, Erice A, Polsky B, Hirsch MS, Owens S, Lamb K: Viability and functional activity of cryopreserved mononuclear cells. Cocktail 1 consisted of CD4 FITC/IFN +IL-2 PE/CD8 PerCP-Cy5.5/CD3 APC. CD4 counting precision has also been shown to be dependent upon the number of events collected, gating, and use of automated analysis [33, 34]. Standardization of cytokine flow cytometry assays. Maecker HT, Dunn HS, Suni MA, Khatamzas E, Pitcher CJ, Bunde T, Persaud N, Trigona W, Fu TM, Sinclair E, Bredt BM, McCune JM, Maino VC, Kern F, Picker LJ: Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. These are detected by the PMT and converted to a voltage pulse, known as an event.
2003, 283 (1-2): 141-153. If you arelooking to get to grips with flow cytometry analysis, check out our free online flow cytometry training. A general guideline for the relative intensities of the various fluorochromes is, from brightest to dimmest, PE, PE-Cy 7, PE-Cy5, APC, APC-Cy7, Alexa Fluor 647, Alexa Fluor 700, FITC, Pacific Blue, Alexa Fluor 488. A set of peptides consisting of epitopes from CMV, EBV, and influenza (CEF) [13] was used as a positive control, due to restrictions on international shipment of SEB. A significant portion of inter-laboratory variability could be eliminated by use of centralized analysis and/or a dynamic gating template. In order to allow more meaningful comparisons between laboratories and prioritization of emerging vaccine candidates, and thereby accelerate HIV vaccine development, this ICS standardization study was undertaken. With the possibility of achieving inter-laboratory C.V.'s of less than 20%, even with relatively low responses, ICS compares favourably to ELISPOT, for which interassay C.V.'s of 1718% for PHA and 5565% for Candida have been reported [29, 30]. However, variability due to individual analysis can be excluded by comparing only centrally analysed data (bottom row of Table 3). The PE channel PMT will detect light emitted from PE (phycoerythrin) at 575 nm wavelength. Sample names are listed along the X axis. While CD4 backgrounds were very similar between experiments, CD8 backgrounds varied. Instrument setup was at the discretion of the individual laboratory, and was either manual (using isotype control stained cells to set PMT voltages, and single-stained cells to set compensation) or automated (using BD FACSComp software and BD Calibrite beads (BD Biosciences)). When centralized automated analysis was applied to the data, backgrounds were not usually reduced. Some of these sites experienced high backgrounds in the negative control wells adjacent to the SEB-containing wells. Cocktail 2 consisted of IFN FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC, and cocktail 3 consisted of IFN FITC/CD69 PE/CD8 PerCP-Cy5.5/CD3 APC. Note that the data points for all three assay types cluster together, indicating that variability is similar for all three assay types. 2004, Wayne, PA , NCCLS, I/LA26-A. The inter-lab C.V.'s for this experiment averaged slightly lower than those for the whole blood experiments (2532% for manual analysis; 2325% for centralized automated analysis). Manual versus automated gating templates. T cell production of IFN, and increasingly also IL-2, is taken as a measure of vaccine immunogenicity in experimental vaccine trials. statement and Forward and side scattered light and fluorescence from stained cells are split into defined wavelengths and channeled by a set of filters and mirrors within the flow cytometer. Flow Cytometry Support CenterFind technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help. With the high viabilities and recoveries obtained for cryopreserved PBMC in this study, there was no obvious relationship between viability/recovery and response. Clin Chem Lab Med. The percent CD69+IFNg+ cells obtained is very similar to that obtained by manual gating in this example, since manual gating was performed so as to include CD3dim and CD8dim cells. Once such a template was created and optimized (see Materials and Methods section for description), it could also have been provided to individual sites in order to yield the same results. 2000, 25 (2): 109-114. There were a significant number of CD8 samples that exhibited high spontaneous cytokine production. Kleeberger CA, Lyles RH, Margolick JB, Rinaldo CR, Phair JP, Giorgi JV: Viability and recovery of peripheral blood mononuclear cells cryopreserved for up to 12 years in a multicenter study.
The use of microtiter plates containing lyophilised reagents simplified the ICS protocol, and appeared to improve assay reproducibility. The results of the second trial are shown in Figure 4. PubMed 2000, 14 (16): 2625-2627. In a long pass dichroic filter, photons above a specific wavelength are transmitted straight ahead, whilst photons below the specific wavelength are reflected at a 90 angle. Blood. The template was then used, without further adjustment, on all the files of a given experiment. The PMTs convert the energy of a photon into an electronic signal a voltage. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Results from each site are indicated as a circle, with median responses for each sample indicated as a horizontal bar.
(C) Control cell data from Experiment 4. 10 million per second or 10 per microsecond). Clin Diagn Lab Immunol. In fact, there was no obvious relationship of viability or recovery with response, perhaps because viabilities and recoveries were virtually all within generally acknowledged limits of acceptability (>80% viability, >50% recovery) [11, 12]. Sheath fluid focuses the cell suspension, causing cells to pass through a laser beam one cell at a time. For example, a minimum number of collected events could be specified (sites in this study were asked to collect 10,00040,000 CD4+ or CD8+ T cells per sample, or 60,000 CD3+ cells). Figure 1. When SEB was replaced with CEF as a positive control, no such problems were noted. Flow Cytometry Learning CenterAccess flow cytometry educational resources for better experiment planning and execution. Provided by the Springer Nature SharedIt content-sharing initiative. The blood was incubated for 6 hours in the presence of brefeldin A, either with no stimulus, Staphylococcal enterotoxin B (SEB), or a mixture of overlapping peptides corresponding to the CMV pp65 protein [810].
The Invitrogen Immunology at Work Resource Center is a learning center with technical content designed for new and experienced life scientists alike exploring the field of immunology. Results from each site are indicated as a circle, with median responses for each sample (105, 1040, and 1090) indicated by a horizontal bar. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Data from experiment 4 are for cocktails 2 and 3 only, to be comparable with experiments 13. This format lends itself to international shipping of reagents (because there is no need for refrigeration), and also to larger clinical trials (because of the stability of the lyophilised reagents). 2003, March: 42-50. Trying to incorporate a reagent into your panel?Ask our technical specialists which you should purchase and how to incorporate into your experiment, Need to add a flow cytometer to your lab?Learn more about the Invitrogen Attune NxT flow cytometer here. This experiment also sought to compare two different types of staining antibody cocktails: the two cocktails used in the previous three experiments (IFN FITC/CD69 PE/CD4 PerCP-Cy5.5/CD3 APC and IFN FITC/CD69 PE/CD8 PerCP-Cy5.5/CD3); and one that combined CD4 and CD8 staining in a single sample, as well as adding IL-2 staining (CD4 FITC/IFN +IL-2 PE/CD8 PerCP-Cy5.5/CD3 APC). (A) Data reported by individual sites. The C.V. for each sample is listed across the top of each panel, along with the mean C.V. for that set of samples. As a positive control in Experiment 4, a set of PBMC were SEB-activated, processed, stained, and then lyophilised in certain wells of the lyophilised antibody plates. These values were determined to be about 8% and 20% C.V., respectively. For Research Use Only. Viabilities and recoveries for cryopreserved PBMC samples used in Experiment 3. The CD8 responses, while positive, were considerably lower than those seen with SEB in the previous experiments.
Replicate cryopreserved vials were sent to each of 7 sites, where they were thawed, rested overnight, stimulated, processed, and analyzed. This is a general pattern; some differences in the relative intensities are seen for individual antibodies. All rights reserved. J Immunol Methods. (A) Schematic of protocol for Experiments 13, performed using liquid antigens and antibodies. Figure 8. 2002, 185 (12): 1709-1716. The post-thaw viability and recovery of the PBMC samples from each site are shown in Figure 5. Dot plot of FS versus SS. These sensors are called photomultiplying tubes (PMTs). Cytokine-positive cells in the absence of stimulus are expressed as a percentage of CD4+ or CD8+ T cells.