18 color lsrii flow cytometer

Splenocytes were stimulated by BARF1 peptides for 5 h with a protein transport inhibitor (Thermo Fisher Scientific). A cell stimulation cocktail and R10, with a protein transport inhibitor, were used as positive and negative controls, respectively. After stimulation, cells were stained with LIVE/DEAD violet (Thermo Fisher Scientific) for viability. CD3e (17A2), CD4 (RM4-5), CD8b (YTS156.7.7), IFN-γ (XMG1.2), TNF-α (MP6-XT22), and IL-2 (JES56-5H4) fluorochrome-conjugated antibodies (all from BioLegend, San Diego, CA, USA) were used for surface and intracellular staining. The samples were run on an 18-color LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by FlowJo v.10.8.1 (BD Biosciences).

After euthanasia, the mouse ears were removed, split in half, and processed into single cell suspensions; as previously reported⁶⁶ . The cells suspensions were blocked with a 1:10 dilution of anti-mouse CD16/32 Fc blocking reagent (eBioscience Cat# 14-0161-82) for 15 minutes at 4 °C. The samples were washed with PBS, then 100 μL of a 1:200 dilution of each antibody was added to each sample for 30 minutes at 4 °C, including: CD11b (Biolegend Cat# 101243), CD3 (eBioscience Cat# 11-0032-82), CD45 (eBioscience Cat# 58-0451-82), GR-1 (eBioscience Cat# 47-5931-82), F4/80 (Invitrogen Cat# 4323732), CD4 (Biolegen Cat# 100424), CD8 (eBioscience Cat# 61-0081-82) and fixable viability dye (Invitrogen Cat # 4339520). Lastly, the samples were washed with PBS and then fixed with 2.5% formalin. The cells were processed through an 18-color LSRII flow cytometer (BD Biosciences) at a flow rate of 35 μL/minute, and the data was analyzed with FlowJo version 8.8.7 (FlowJo LLC, Ashland, OR). Appropriate compensation controls and fluorescence minus one samples were used for calibrating the machine and gating the samples. All flow cytometry data was converted to total cell counts per ear, using total cell counts obtained by manually counting the cells in each sample with a hemocytometer.

Cells were stained with 10 µM carboxymethyl-H₂-dichlorofluorescein diacetate (CM-H₂DCFDA, Molecular Probes), 5 µM dihydroethidium (DHE, Molecular Probes), or 5 µM MitoSOX Red (Molecular Probes) in 1× HBSS for 15 min at 37 °C. Using a BD LSRII 18-color flow cytometer (DCF excitation 488 nm, emission 515 nm; DHE and MitoSOX excitation 532 nm, emission 575 nm), 10,000 events gated as singlets were collected for analysis. FlowJo software was used to determine mean fluorescence intensity.

Cells were stained with 50 nM MitoTracker Green FM (Molecular Probes, Life Technologies, Eugene, OR) in 1× HBSS for 30 min at 37 °C. Using a BD LSRII 18-color flow cytometer (excitation 488 nm, emission 515 nm), 10,000 events gated as singlets (FSC-A/SSC-A then SSC-H/SSC-W) were collected for analysis. FlowJo software (FlowJo, Ashland, OR) was used to determine mean fluorescence intensity.