Ebioscience intracellular fixation permeabilisation buffer set

B cells were isolated from PBMC using B Cell Isolation Kit II, human (Miltenyi Biotec) and T cells from an allogenic donor were isolated using CD2 MicroBeads, human (Miltenyi Biotec) and cell isolation was performed using the Miltenyi Biotec MidiMACS™ separator following the manufacturer’s instructions. B cells were cultured with 5-OP-RU at indicated concentrations for 4 hr. The B cells were then washed twice prior to addition of BFA and allogenic (MHC miss-matched, as MR1 is monomorphic) T cells at 1:1 effector:target ratio and cultured for 18 hr. Cells were collected and stained with viability dye, prior to fixation and permeabilisation using the eBioscience™ Intracellular Fixation & Permeabilisation Buffer Set (Invitrogen). Antibodies diluted in permeabilisation buffer were then incubated for 45 min at room temperature.

PBMC were cultured and pulsed with a range of stimulants. For cytokine stimulation: 100 U/mL human interleukin (IL)-2 (Roche), 200 ng/mL human IL-12 (Miltenyi Biotec) and 200 ng/mL human IL-18 (Biolegend), two doses 72 hr apart, with brefeldin A (BFA) (Biolegend) added after second dose and cultured for 18 hr. For bacterial stimulation: E. coli at multiplicity of infection (MOI) 50 was added to PBMC, left for 1 hr prior to adding 50 μg/mL gentamicin (Sigma) and BFA and cultured for 18 hr. For (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) stimulation: 10 ng/mL of HMBPP (Cayman Chemicals) was added and cultured for 4 hr before adding BFA and cultured for 18 hr. For a positive control: 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) and 1 µg/mL ionomycin (Sigma) were added with BFA and cultured for 18 hr. Following stimulation, cells were collected and stained with viability dye and stained with surface antibodies for 20 min on ice, prior to fixation and permeabilisation using the eBioscience™ Intracellular Fixation & Permeabilisation Buffer Set (Invitrogen). Intracellular antibodies were then incubated for 45 min diluted in permeabilisation buffer.

PBMC were cultured with 1 nM 5-OP-RU for 4 hr, then Brefeldin A solution (eBioscience) was added and cells cultured for 18 hr. Cells were collected and stained with viability dye, prior to fixation and permeabilisation using the eBioscience™ Intracellular Fixation & Permeabilisation Buffer Set (Invitrogen). Antibodies diluted in permeabilisation buffer were then incubated with cells for 45 min at room temperature.