Fixation and permeabilization buffer

Mice were euthanized by cervical dislocation after sodium pentobarbital (50 mg/kg, i.p.) anesthesia every 4 h in the initial 24 h after LPS injection. Peritoneal cells were harvested by peritoneal lavage using 10 ml PBS and the total numbers of cells were counted. Erythrocytes were removed by cell lysis in FACS lysing solution (BD Biosciences, Heidelberg, Germany). Peritoneal cells and spleen cells were pre-incubated for 10 min at room temperature in 10% FCS to block Fc receptors and non-specific binding. After washing three times, cells were stained with Abs specific for mouse CD3, NKp46, Tim-3, CD69, CD107a, CD4, CD8, CD11b, CD11c, F4/80, galectin-9 and incubated on ice for 30 min. For intracellular staining, the cells collected after surface staining were fixed and permeabilized with Fixation and Permeabilization Buffer (BD Pharmingen). After permeabilization, cells were stained with Abs to IFN-γ, granzyme B, perforin and analyzed using a FACScan flow cytometer (Becton Dickinson). NK cell apoptosis was assessed using an Annexin V-PI Apoptosis Detection Kit according to the manufacturer’s instructions. Data analysis was performed using FlowJo version 7.6.1 software (TreeStar).

Cell proliferative capacity was detected using the kFlour647 ClickiT EdU Flow Detection Kit (KeyGEN BioTECH) (Zhang et al., 2021 (link)). AML cells were seeded in 6-well plates and cultured for 48 h with or without GSK269962A. Cells were then incubated with 20 μM EdU for 4 h after drug exposure, after which they were washed three times and then treated with fixation and permeabilization buffer (BD Biosciences) for 30 min. Next, cells were washed and incubated with 200 μl of iClick reaction solution for 30 min. The EdU-positive rate was measured by flow cytometry and was analyzed by the FlowJo software.

Sh-V0a2 transfected/untransfected cells (2.5 x10⁵ cells/tube) were washed with HBSS containing 0.1% FBS. For surface staining, the cells were incubated with mouse monoclonal FasL or Fas antibody conjugated to A₆₄₇ or A₄₈₈ (Covance, Denver, PA) in PBS for 40 min at RT. For the intracellular staining, the cells were fixed and permeabilized using fixation and permeabilization buffer (BD Biosciences, San Jose, CA, USA) and the cells were stained as described above. For the indirect staining, the cells were incubated with unconjugated antibodies (caspase-8) for 1 h at RT and subsequently washed twice with PBS and then stained with conjugated secondary antibody (Abcam, USA) for 30 min at RT. Appropriate isotype and unstained controls were used for the experiments. The stained cells were analyzed on a BD LSR II flow cytometer with FlowJo software (Tree Star). Experiments were performed at least twice in duplicate.

The cells were harvested and washed twice with phosphate-buffered saline buffer containing 0.1% bovine serum albumin and 0.05% sodium azide. The expression of surface and intracellular markers was analysed by immunostaining PBMCs with the antibodies against CD3, CD4, CD25, FoxP3, CD161, IL-23R; (BD PharMingen, USA). For surface staining, cells were incubated with the relevant antibodies at 4 °C in the dark for 30 min. For detection of intracellular FoxP3, CD4CD25 cells were fixed with fixation and permeabilization buffer (BD Bioscience PharMingen, USA) and stained according to permeabilization/fixation Kit protocol. The doublets were excluded; by opting height versus width FSC gating in all the flow cytometry experiments before sample acquisition (Fig. 1a). Suitable fluorescence minus one (FMO) controls was used to define the negative population in all the experiments. The stained cells were then analyzed by flow cytometry (FACS, ARIA III, BD Biosciences, USA). Fluorescence profiles were analyzed using FlowJo software (BD Biosciences). The results expressed as a percentage of positive cells.