Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2003 Sep 2:4:9.
doi: 10.1186/1471-2172-4-9. Epub 2003 Sep 2.

Performance of plate-based cytokine flow cytometry with automated data analysis

Maria A Suni  1 , Holli S Dunn, Patricia L Orr, Rian de Laat, Elizabeth Sinclair, Smita A Ghanekar, Barry M Bredt, John F Dunne, Vernon C Maino, Holden T Maecker
Affiliations
Comparative Study

Performance of plate-based cytokine flow cytometry with automated data analysis

Maria A Suni et al. BMC Immunol. .

Abstract

Background: Cytokine flow cytometry (CFC) provides a multiparameter alternative to ELISPOT assays for rapid quantitation of antigen-specific T cells. To increase the throughput of CFC assays, we have optimized methods for stimulating, staining, and acquiring whole blood or PBMC samples in 96-well or 24-well plates.

Results: We have developed a protocol for whole blood stimulation and processing in deep-well 24- or 96-well plates, and fresh or cryopreserved peripheral blood mononuclear cell (PBMC) stimulation and processing in conventional 96-well round-bottom plates. Samples from both HIV-1-seronegative and HIV-1-seropositive donors were tested. We show that the percent response, staining intensity, and cell recovery are comparable to stimulation and processing in tubes using traditional methods. We also show the equivalence of automated gating templates to manual gating for CFC data analysis.

Conclusion: When combined with flow cytometry analysis using an automated plate loader and an automated analysis algorithm, these plate-based methods provide a higher throughput platform for CFC, as well as reducing operator-induced variability. These factors will be important for processing the numbers of samples required in large clinical trials, and for epitope mapping of patient responses.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Representative examples of tube- and plate-based CFC results. (A) Whole blood from a CMV seropositive donor was stimulated (or not) with CMV pp65 peptide mix in 15 ml conical polypropylene tubes (top panels) or a deep-well 96-well polypropylene plate (bottom panels). (B) PBMC from another CMV seropositive donor were stimulated as above in 15 ml conical polypropylene tubes (top panels) or a 96-well round-bottom tissue culture plate (bottom panels). (C) Whole blood from an HIV-seropositve donor was stimulated (or not) with HIV p55 gag peptide mix in 15 ml conical polypropylene tubes (top panels) or a deep-well 24-well round-bottom polypropylene plate (bottom panels). Backgrounds and response to peptide mix were essentially equivalent in tubes and plates in each case. All data are gated on CD3+CD8+ cells.
Figure 2
Figure 2
Correlation of tube- and plate-based CFC assays. Stimulation vessels and antigens were as in Figure 1. (A) Correlation of whole blood CMV and SEB responses in 17 CMV seropositive donors, done in tubes versus deep-well 96-well conical polypropylene plates. (B) Correlation of PBMC CMV and SEB responses in 13 CMV seropositive donors, done in tubes versus 96-well round-bottom tissue culture plates. (C) Correlation of whole blood HIV and SEB responses in 10 HIV seropositive donors, done in tubes versus deep-well 24-well round-bottom polypropylene plates.
Figure 3
Figure 3
Mean fluorescence intensity of IFNγ staining using tube- or plate-based CFC. Data are taken from the samples of Figure 2. Error bars represent SEM.
Figure 4
Figure 4
Effect of overnight "resting" on cryopreserved PBMC. (A) Representative comparison of fresh PBMC, cryopreserved PBMC, and cryopreserved PBMC incubated overnight prior to stimulation (all from the same CMV+ donor). Unstimulated samples (top row) and CMV pp65 peptide mix-stimulated samples (bottom row) show similar results, but the IFNγ fluorescence intensity (MFI) is greatest in the cells rested overnight prior to stimulation. (B) Results of triplicate samples stimulated with CMV pp65 peptide mix and analyzed for percentage of IFNγ+ cells or IFNγ mean fluorescence intensity. Error bars represent SEM. All data were gated on CD3+CD4- cells. Similar results were obtained for TNFα+ cells (not shown). Results are representative of two similar experiments.
Figure 5
Figure 5
Cell recovery in tube- and plate-based CFC assays. Recovery was calculated by addition of a known number of beads to each CFC sample at the end of sample processing. Data are shown as a percentage of cells counted in fresh whole blood or PBMC from the same donor. Error bars represent SEM of 3 donors for each assay.
Figure 6
Figure 6
Automated gating of CFC samples. (A) Example of automated gating. A cluster-finding algorithm is employed to automatically identify small lymphocytes in forward versus side scatter (R1, top left panel; this region can also be drawn manually during sample acquisition). A similar automated region is calculated for CD3+CD8- and CD3+CD8+ lymphocytes (R2 and R3, top right panel). R2 and R3 are set for maximal size to allow inclusion of activated cells that have down-modulated CD3 and CD8 (bold dots). The lower left plot, gated on regions R1 and R2 (CD3+CD8- lymphocytes), uses the cluster-finding algorithm to identify the CD69-IFNγ- population (R4). This population is tethered to a rectangular region that identifies the CD69+IFNγ+ cells (R5). The lower left plot is similar, but gated on R1 and R3 (CD3+CD8+ lymphocytes). The percentage of gated events in R5 is reported. (B) Correlation of the automated gating template to expert manual gating. Whole blood from 23 CMV seropositive donors was stimulated in deep-well 96-well plates, then analyzed manually or using the automated template in (A). Using a batch analysis protocol, many CFC samples can be analyzed and the resulting data downloaded to a spreadsheet in a rapid fashion.
Figure 7
Figure 7
Automated gating of HIV-1-seropositive CFC samples. (A) Correlation of automated gating to expert manual gating. Whole blood from 39 HIV-1-seropositive donors was stimulated using the antigens shown, then analyzed manually or with the automated template of Figure 6A. (B) Revised template designed for samples with very low numbers of CD4+ cells. A fixed region for CD3+CD4+ cells (R3) has been created and tethered to the automated region R2. (C) Improved performance of the revised template for calculating CD4+ responses from the dataset of (A). Note that the slope of the linear regression line for pp65 peptide mix and SEB stimulation is now closer to 1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ghanekar SA, Maecker H. Cytokine flow cytometry: multiparameter approach to immune function analysis. Cytotherapy. 2003;5:1–6. doi: 10.1080/14653240310000029. - DOI - PubMed
    1. Maecker HT, Maino VC. Flow cytometric analysis of cytokines. In: Hooks J, editor. Manual of Clinical Laboratory Immunology, 6th ed. Washington, DC, ASM Press; 2002.
    1. Maecker HT, Maino VC, Picker LJ. Immunofluorescence analysis of T-cell responses in health and disease. J Clin Immunol. 2000;20:391–399. doi: 10.1023/A:1026403724413. - DOI - PubMed
    1. Shacklett BL. Beyond 51Cr release: new methods for assessing HIV-1-specific CD8+ T cell responses in peripheral blood and mucosal tissues. Clin Exp Immunol. 2002;130:172–182. doi: 10.1046/j.1365-2249.2002.01981.x. - DOI - PMC - PubMed
    1. Suni MA, Picker LJ, Maino VC. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J Immunol Methods. 1998;212:89–98. doi: 10.1016/S0022-1759(98)00004-0. - DOI - PubMed

Publication types

LinkOut - more resources

-