S1A and S1C). viSNE analysis effectively characterized PBMC using eight features per cell and identified comparable frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of unsupervised analysis programs and extended multiparameter cytometry will be Papain Inhibitor indispensable tools for detecting perturbations in protein expression in both health and disease. phenotyping or phenotyping following 16 hours of SEB (EMD Millipore, Billerica, MA) stimulation, and then pelleted again before resuspension in room temperature PBS (staining were further divided among flow cytometry tubes (Falcon 2052, BD-Biosciences, San Jose, CA) for fluorescence or mass cytometry staining, described below. Cells for culture were stimulated by addition of SEB to achieve a final concentration of 1 1 g/mL in 200uL of 10 106 cells/mL in 48-well flat bottom culture plates (Costar, Corning Incorporated, Corning, NY, USA). After 16 hours of incubation at 37C in a 5% CO2 incubator, cells were removed from the plate, washed twice in PBS, and stained as described below. Fluorescence cytometry For each healthy donor, 2 106 PBMC were stained in 200L PBS. PBMC were incubated first with a viability dye for 10 minutes (LIVE/DEAD Aqua, Life Technologies), washed once in PBS, and then stained with combinations of fluorescently-tagged antibodies (Table 1). For phenotyping, cells were stained with Panels 1C5 from Table 1 (for antibody information see Table S1). For phenotyping following stimulation, cells were stained with Panels 3C5 from Table 1 at 16 hours after addition of SEB. After staining, all cells were washed twice in PBS and fixed with 2% paraformaldehyde (PFA, Electron Microscopy Services, Fort Washington, PA, USA) and refrigerated up to 24 hours until analysis around the Special Order Research Product (SORP) BD LSRFortessa (BD Biosciences, San Jose, CA) at the Vanderbilt Flow Cytometry Shared Resource. Table 1 Fluorescence cytometry instrument and antibody panel information data was used (antibodies solely used in Panel 1 or 2 2, Table 1); N was 12 when an and value were used from each subject. Results Fluorescence and mass cytometry panels to track T cell identity Panel design Five fluorescence cytometry panels currently in use in our laboratory were used to measure 20 well-established cell surface markers chosen to provide a systematic view of T cell activation after SEB stimulation (Table 1). Fluorochrome and antibody conjugates were chosen based upon current availability in the laboratory and their compatibility with the BD LSRFortessa at the Vanderbilt Flow Cytometry Shared Resource. A single mass cytometry panel was developed to measure the same set of 20 surface markers captured by the five fluorescence cytometry panels (Table 2). While mass cytometry avoids the severity of channel overlap that affects fluorescence cytometry, crosstalk between channels exists. Crosstalk leads to false signals and must be taken into consideration when designing a panel for mass cytometry and gating cellular populations. The three sources of crosstalk result from variations in abundance sensitivity, isotope purity, and oxide formation (Fluidigm.com Maxpar Panel Designer Papain Inhibitor User Guide). These types of crosstalk can contribute to signal in the M1 and M+16 masses from the dominant signal at mass M. To minimize Papain Inhibitor crosstalk within this panel, four of the 20 antibodies were custom conjugated to metals (Table 2, bolded). Antibody titrations Single antibody titrations were performed for all those fluorochrome-conjugated antibodies (FCAs) and metal-conjugated antibodies (MCAs) as needed. As an Papain Inhibitor example, Physique S1 shows the titration Mouse monoclonal to COX4I1 of CD4-Nd145 and CD4-PETR (PE-Texas Red). CD4-Nd145 was titrated from 0uL to 0.5uL (recommended amount) with DNA intercalator to identify single cells for analysis (Fig. S1A). The mean mass intensity (MMI) of the CD4-Nd145+ population shifted from 6.63 to 264.48 while the MMI of the CD4-Nd145-population stayed between ?0.40 and ?0.19 (Fig. S1A and S1C). The standard deviation of the CD4-Nd145- population was always below 1 (Fig. S1A and S1C). With increasing antibody concentrations the frequencies of the CD4-Nd145+ populations increased from 0.18% to 60.73% and stain index values increased from 11.78 to 149.16 (Fig. S1A and S1C). A single antibody titration was also performed with CD4-PETR from 0uL to 2uL and FSC and SSA properties were used to identify single cells for analysis (Fig. S1B). The mean fluorescence intensity (MFI) of the.
S1A and S1C)
