Foxp3 fix perm buffer

The following directly labeled anti-mouse monoclonal antibodies were used for BM cell phenotypical analysis: CD90.2-APC and CD45-PE/Cy7 for lymphoid progenitors, CD61-APC and CD41-FITC for megakaryocytic population, CD71-PE and Ter119-FITC for erythroid precursors, CD11b-PE and Gr1-FITC for granulocytes/monocytes progenitors, Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-FITC, Sca1-PE, cKit (CD117)-APC for hematopoietic stem cells, all purchased from BioLegend (BioLegend, San Diego, CA, USA).
The phenotypical analysis of splenocytes was performed using the following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE, I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was used.
Single-cell suspensions of splenocytes or BM cells were incubated with the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C for 20 min for cell surface staining. For intracellular staining (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer (eBioscience), according to the manufacturer’s instructions. Measurements were performed with a FACSCalibur flow cytometer as described above.

Labeled and purified antibodies were purchased from BD Biosciences, BioLegend, or eBioscience (Table 1). Single cell suspensions were incubated sequentially with anti-CD16/32 (to block Fc receptors) then primary antibodies. Data were collected on a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star). For intracellular cytokine analysis, 5–10 × 10⁵ leukocytes were restimulated with ionomycin (1 µg/ml) and PMA (25 ng/ml) in the presence of Brefeldin A (10 µg/ml) for 4.5–5 h at 37°C. Following staining for surface antigens, cells were stained for intracellular cytokines using a Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer’s instructions. For short-term pulse BrdU labeling, mice were injected intraperitoneally with 1 mg/mouse BrdU (Sigma-Aldrich) in 100 µl PBS 1 h before sacrifice. BrdU incorporation was examined using a BrdU flow kit (BD Biosciences), according to the manufacturer’s instructions. For the intracellular staining of Foxp3 and Ki-67, 1 × 10⁶ leukocytes were stained for surface antigens and then were fixed and permeabilized with Foxp3 Fix/Perm Buffer (eBioscience) according to the manufacturer’s instructions.