Anti mouse cd4 af700

Thymocytes and splenocytes isolated from three-month-old male C57BL/6 J mice (MARP) were seeded at 1 × 10⁶ cells per well in RPMI supplemented with 2 mM L-glutamine, 1 mM MEM sodium pyruvate, 100 uM MEM non-essential amino acids, 5 mM HEPES buffer solution, 55 uM 2-mercaptoethanol, 10 U/mL IL-2, 100 U/mL penicillin and 100 ug/mL streptomycin. Cells were exposed to either purified TcdB from strain VPI10463 (Abcam) diluted to 80 ng/mL, 16 ng/mL, 3.2 ng/mL, 0.64 ng/mL and 0.128 ng/mL, or 10% DMSO or 1 μM oligomycin (as high or moderate positive controls to induce cell death in leukocytes), for 20 hours, using untreated cells as a control, before imaging cells on an EVOS FL Auto Cell Imaging System (Invitrogen). Following cell imaging, the cell surfaces were stained with LIVE/DEAD™ Fixable Aqua (1:400; Invitrogen), Propidium Iodide (1:800; BD Biosciences), anti-mouse CD3-PE (1:400; BD Biosciences), anti-mouse CD4-AF700 (1:400; Biolegend), and anti-mouse CD8-PacBlue (1:400; BD Biosciences) antibodies for 15 minutes, in the dark, on ice. Cells were washed three times, before resuspending in 200 µL of FACS buffer, and quantified using a BD LSRII Flow Cytometer (BD Biosciences). Analysis was performed on FlowJo version 9.9.6.

After restimulation, cells were transferred to a V-bottom 96-well plate (Greiner Bio-One, #651180) and washed once with PBS. Cells were resuspended in TruStain Fc blocking solution (BioLegend, #101320) for 10 min at room temperature. Afterwards, cells were incubated with anti-mouse CD4-AF700 (BioLegend, #100429, 1/200 dilution), anti-mouse CD8b-AF488 (BioLegend, #126627, 1/200 dilution) and Zombie Aqua fixable viability dye (BioLegend, #423101, 1/200 dilution) diluted in PBS for 20 min on ice. Cells were washed once with PBS and fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, #15710) for 15 min on ice. After fixation, cells were washed once with PBS and permeabilized using the intracellular staining permeabilization wash buffer (BioLegend, #421002) following manufacturer’s instructions. Cell suspensions were then incubated with anti-mouse IFNγ-APC (BioLegend, #505305, 1/200 dilution) and TNFα-PE (BioLegend, #506305, 1/200 dilution) diluted in intracellular staining permeabilization wash buffer for 30 min at room temperature. Cells were washed twice with the permeabilization wash buffer, resuspended in 100 μL PBS and transferred to FACS tubes. Fluorescence intensities and the percentage of IFNγ- and TNFα-expressing cells were measured using an LSRII (BD Biosciences) flow cytometer. Data analysis was performed with the FlowJo V10 software (Tree Star).