Flow Cytometer Setup and Data Acquisition
Flow cytometric data were acquired using a BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA). This instrument is an air-cooled 4-laser benchtop flow cytometer with the ability to detect up to 12 individual fluorescent signals. The lasers and filters used were supplied with the instrument from BD Biosciences. The lasers used in this study were the following: solid state, 50 mW, 488 nm (blue); solid state, 50 mW, 405 nm (violet); solid state, 150 mW, 532 nm (green); helium-neon, 70 mW, 633 nm (red). The photomultiplier (PMT) setup is depicted in Additional File 3 Figure S2 and used a trigon configuration for blue, violet, and red laser paths and an octagon configuration for the green detector arrays. Dichroic long pass mirrors were used on the inner rings, and band pass filters used on outer rings. A 2-Blue, 2-Violet, 5-Green, 3-Red fluorochrome excitation configuration was used in this study (Additional File 3 Figure S2). Instrument performance was validated using BD Cytometer Setup and Tracking (CS&T) beads (part #: 910723, Lot ID: 75445, BD Biosciences, San Jose, CA). This validation was performed routinely and prior to each of the large phenotyping experiments. Triggering was set on the forward scatter (FSC) signal. FACS DIVA 6.0 software (BD Biosciences) was used to acquire digital data.
Fluorochrome Compensations and PMT Settings
Although PMT settings and compensations should be adjusted for each instrument and stain setup, we include here the settings used for our instrument which were the following: the FSC setting included both area and width, with a threshold of 10,000, and a PMT Voltage of 315. The SSC setting included area with a PMT voltage of 300. All other PMT settings (log scale) were as follows: Pacific Blue = 380, APC = 545, APC-Cy7 = 568, FITC = 539, PerCP = 642, PE = 577, PE-Texas Red = 501, PE-Cy5.5 = 479, PE-Cy7 = 551, and Pacific Orange = 468. The PMT voltage settings were manually set using unstained cells as a reference. Each histogram was adjusted to allow a full view of the peak at approximately the same intensity for each fluorochrome.
Unstained and single fluorochrome-mAb stained thymocytes were used to establish baseline instrument application settings and compensation settings for each fluorochrome measured. To determine and validate compensation settings for fluorescence on small populations, cells of interest were sort enriched prior to use. Preliminary experiments were designed to first assess the staining patterns of small panels of mAbs and then the staining patterns observed in these smaller panels were compared with those previously observed in simple two-, three-, or four-color experiments. After comparable results were obtained, an additional interest marker was added, and separate "fluorescence minus one" (FMO) controls were prepared to ensure that the staining patterns and all subset populations of the original markers were not changed by the addition of the new marker. FMO control histograms are shown in Additional File 4 Figure S3. Using this sequence, the 10-color staining panel was eventually successfully validated.
For the pSTAT assays shown in Figure 3B, the cell subsets indicated in the figure legend were sort-purified using a two-laser Moflo cell sorter (Beckman-Coulter, Fullerton, CA) and Summit v4.3 software. For the validation of compensation settings for the small DN thymocyte subsets, cells were stained with a single fluorochrome and sort-enriched using the MoFlo cell sorter prior to use on the LSR II. These cells were stained and positively enriched using one of the following mAb or mAb combinations: cKit-APC, CD25-PerCP, CD44-APC-Cy7, or Lin-biotin/TCR γδ-biotin followed by streptavidin-PE-Texas Red.
Data were analyzed using FACS DIVA 6.0 software (BD Biosciences). For all experiments, cell doublets and clusters were gated from the analysis using doublet discrimination (FSC width versus FSC area). Cell debris and small particles were excluded by gating out events with low forward scatter. The gating strategy is shown in Figure 2. CD4 and CD8 double positive and single positive populations that were negative for the non-T lineage markers (Lin) and TCR-γδ were further discriminated as follows: DP (CD4+CD8+), SP4 (TCR+CD4+CD8-), and SP8 (TCR+CD4-CD8+). DP TCR negative, DP TCR intermediate, and DP TCR high were separated by TCR-β chain (H57-597) expression. CD4-CD8- (DN) populations were further defined as follows: DN1 (Lin-CD25-CD44+), DN2 (Lin-CD25+CD44+), DN3 (Lin-CD25+CD44-) and DN4 (Lin-CD25-CD44-). Five subsets of TCR negative DN1 thymocytes (DN1a-DN1e), as defined by c-Kit and CD24 expression were discriminated as previously demonstrated . DN3 and DN4 were also further defined based on CD25 and CD28 expression. DN3 cells were divided into CD28 low/CD25 high (DN3a), CD28 intermediate/CD25 high (DN3b) and CD28 intermediate/CD25 intermediate (DN3c). DN4 cells were divided into CD28 intermediate (DN4a), CD28 high (DN4b), and CD28 low (DN4c) cells as described in the results section.
In Figure 3, median fluorescent intensity (MFI) ratios were calculated based on the FSC-adjusted MFI values of the indicated specific cytokine receptor stains divided by the FSC-adjusted MFI values from the corresponding isotype control stains. FSC adjustment (PE MFI/FSC MFI) was done in order to account for cell size differences in the different subsets. This also allowed for normalization across the multiple experiments. MFI ratio averages and standard deviations were calculated from a minimum of five separate experiments for each cytokine receptor. To do overlay plots, FCS data were exported, and analyzed by FlowJo (Tree Star, Inc, Ashland, OR). Statistical analysis was generated using GraphPad Prism v. 4.00 (La Jolla, CA).
Mice used in this study were C57BL/6 females ranging from 6 to 8 weeks of age that were bred and housed at U.S.D.A. approved animal facilities located at the University of Oklahoma in Tulsa and the University of Tulsa. Animal care and all animal experiments were done in accordance with procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council). Protocols were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Oklahoma Health Sciences Center and the University of Tulsa.
Tissue harvest and cell staining
Thymuses were harvested and placed into complete tumor media (CTM) as previously described . Thymuses were pressed through 70-μm nylon screens to generate single thymocyte suspensions . Cells were treated with RBC lysis buffer (Sigma-Aldrich, St. Louis, MO), washed into CTM and counted using a hemocytometer. Cells were incubated with mAb against mouse CD16/CD32 (Fc Block, BD Biosciences) to block potential Fc-mediated binding and then stained at a density of 1 × 108 cell/ml with primary mAbs as indicated in Additional File 5 Table S2 for 45 minutes at 4°C in the dark. After two washes, the cells were further stained with Streptavidin PE-Texas Red (1:1600) for 30 minutes at 4°C in the dark. The monoclonal antibodies used in the 10-color stain experiments were individually tested. Optimal antibody titers (Additional File 5 Table S2) were determined for each monoclonal antibody and/or their secondary stain (Streptavidin PE-Texas Red) by staining thymocytes with mAb titers ranging from 1:50 to 1:1600, depending on the fluorochrome. For Annexin-V (Biolegend) staining, cells were washed twice after surface marker staining, then resuspended in Annexin-V binding buffer (Biolegend containing Annexin-V-FITC (1:100), and incubated for 15 minutes at room temperature in the dark.
STAT phosphorylation assays
Assessment of IL-6-induced STAT-1 phosphorylation began with surface staining of total thymocytes with anti-CD4, CD8, and CD24 mAbs (Additional File 5 Table S2). These cells were sort-purified into the following populations: CD24loSP4, CD24hiSP4, CD24loSP8, and CD24hiSP8. Sorted cells were then incubated in the presence or absence of 25 ng/ml IL-6 (R&D Systems, Minneapolis, MN) for 20 minutes at 37°C. Intracellular stains were performed using Invitrogen/Caltag Fix & Perm reagents with an additional methanol step as previously described. STAT-1 phosphorylation status was assessed with PE-conjugated anti-pSTAT1 (pY701) (BD Biosciences) using a PE-conjugated mouse IgG2a (BD Biosciences) as an isotype control.