Foxp3 fjk 16s staining kit

Multicolor flow-cytometric analysis of multilineage macrochimerism and Vβ-subunit expression were performed as described previously.5 (link), 13 (link) Chimerism was calculated as the net percentage of donor MHC Class I⁺ (H-2D^d, 34-2-12) cells among specific leukocyte lineages, as described previously.5 (link), 13 (link) Mice were considered chimeric if donor cells were detectable by flow cytometry within both the myeloid lineage and at least one lymphoid lineage. For analysis of Treg phenotype, MAbs with specificity against CD4 (RM4-4) and CD25 (7D4) were used. For intracellular staining, a FoxP3 (FJK-16s) staining kit (eBioscience) was used according to the manufacturer’s protocol. Propidium iodide (PI) was used for dead cell exclusion when appropriate. Surface staining was performed according to standard procedures and flow-cytometric analysis was done on a Coulter Cytomics FC 500 System using CXP software (Coulter, Austria) for acquisition and analysis.

Multicolor flow cytometric analysis of multilineage chimerism was performed as described previously [23 (link), 24 (link)]. Briefly, chimerism was calculated as the net percentage of donor MHC class I⁺ (H-2D^d, 34-2-12) cells among specific leukocyte lineages (CD4⁺ and CD8⁺ T cells, B220⁺ B cells, and Mac1⁺ myeloid cells) [23 (link), 24 (link)]. Mice were considered chimeric if donor cells were detectable by flow cytometry within both the myeloid lineage and at least one lymphoid lineage. For analysis of Tregs mAbs with specificity against CD4 (RM4-4) and CD25 (7D4) were used. For intracellular staining a FoxP3 (FJK-16s) staining kit (eBioscience) was used according to the manufacture's protocol. PI was used for dead cell exclusion when appropriate. Surface staining was performed according to standard procedures and flow cytometric analysis was done on Coulter Cytomics FC500 using CXP software (Coulter, Austria) for acquisition and analysis.

Multicolor flow cytometric analysis of Tregs was performed as described previously [17 (link)]. Monoclonal antibodies (mAbs) with specificity against CD4 (RM4-4), CD25 (7D4), CD62L (Mel-14), and CTLA4 (UC10-4F10-11) were used. For intracellular staining, FoxP3 (FJK-16s) Staining Kit (eBioscience) was used according to the manufacturer's protocol. PI was used for dead cell exclusion when appropriate. Surface staining was performed according to standard procedures, and flow cytometric analysis was done on Coulter Cytomics FC500 using CXP software (Coulter, Austria) for acquisition and analysis.