Rat anti foxp3 fjk 16s

Murine splenocytes or pLN were isolated as described above. Isolated leukocytes were resuspended in complete RPMI medium (10% fetal bovine serum (FBS) in RPMI medium) containing 0, 1, 10 or 100 ng/ml murine IL-2 (Peprotech). Stimulation was performed at 37 °C for 30 mins. Stimulation was terminated by the addition of 1X BD Phosflow^TM Lyse/Fix Buffer (BD Bioscience) at 37 °C for 10 mins. Permeabilization was performed using Perm Buffer III (BD Bioscience) on ice for 30 mins. Cells were then washed 3 times with staining buffer to completely remove traces of Perm Buffer III before proceeding to stain with rat anti-CD4 (RM4–5, BD Bioscience), rat anti-CD25 (7D4, eBioscience), rat anti-Foxp3 (FJK-16s, eBioscience) and anti-pSTAT5 (pY694) (BD Bioscience) antibodies for 1 hr. Human peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy donors using Ficoll-Plaque (GE Healthcare)67 (link). Stimulation of PBMCs was done as above except that human IL-2 (Peprotech) was used and stimulation was done at 37 °C for 15 mins instead. Staining was done using mouse anti-human CD3 (UCHT1, BD Bioscience), mouse anti-human CD4 (SK3, BD Bioscience), mouse anti-human CD45RA (HI100, eBioscience), rat anti-human Foxp3 (PCH101, BD Bioscience) and mouse anti-pSTAT5 (pY694, BD Bioscience) antibodies for 1 hr.

Fifteen microliters of blood were collected from the tail vein. Briefly, blood was lysed using 1X Mouse Lyse Buffer (R&D Systems) and washed twice with PBS. Cells were stained with rat anti-CD4 (clone RM4–5, BD Biosciences), rat anti-CD25 (PC61.5, eBioscience), rat anti-CD44 (IM7, eBioscience) antibodies. Intracellular staining of Foxp3 was done using Foxp3 staining buffer set (eBioscience) and rat anti-Foxp3 (FJK-16s, eBioscience) following manufacturer’s instructions. Data was acquired using BD LSRFortessa™ cell analyzer. Dead cells and duplets were excluded in all analyzes using forward and side scatter gating. Results were analyzed with FlowJo version X software (Tree Star, Inc).