Cd44 pe cy7 im7

Spleen and thymus of six-week-old NOD.Foxp3-GFP/cre (Stock No: 008694, Jackson Laboratory) were processed using frosted glass slides and passed through a 40-micron filter to create single cell suspensions. Red blood cells (RBC) were lysed with ammonium-chloride-potassium buffer prior to staining for flow cytometry. Samples were stained with Fixable Live/Dead Near IR (Invitrogen), washed once with stain buffer (PBS + 2% FBS + 0.05% NaN₃), and Fc receptors were blocked with anti-CD16/32 for 5 minutes at 4°C (Clone 2.4G2, BD Biosciences). Samples were stained for 30 minutes at 4°C with the following anti-mouse antibodies: CD3e-Brilliant Violet (BV) 605 (145-2C11, BioLegend), CD4-PerCP/Cy5.5 (RM4-5, Thermo Fisher Scientific), CD8a-BV711 (53-6.7, BioLegend), CD44-PE-Cy7 (IM7, BioLegend), CD62L-APC (MEL-14, BioLegend), and CD221-PE (3B7, Santa Cruz Biotechnology). Samples were washed once with stain buffer prior to data acquisition on an LSRFortessa (BD Biosciences) and analysis with FlowJo (v10.6.1; Tree Star).

Single cell suspensions of splenocytes were generated from 6-7-week-old pre-diabetic NOD.Foxp3-GFP/cre mice (Stock No: 008694, Jackson Laboratory), followed by RBC lysis. Cells were resuspended at 10⁶/mL in cRPMI and treated with 10 IU/mL rh-IL-2 (Teceleukin) and/or 100 ng/mL rhIGF1 (BioVision) for two days at 37°C. Live/Dead fixable near-IR dead cell stain kit (Invitrogen) was applied according to manufacturer’s instructions for dead cell exclusion. Cells were washed once with stain buffer and then, incubated with anti-CD16/32 (Clone 2.4G2, BD Biosciences) for 5 minutes at 4°C. Samples were stained with the following fluorescently-labeled anti-mouse antibodies at 4°C for 30 minutes: CD4-PerCP-Cy5.5 (RM4-5, eBioscience), CD62L-BV510 (MEL-14, BioLegend), CD44-PE-Cy7 (IM7, BioLegend), CD221-PE (3B7, Santa Cruz Biotechnology), CD25-AF700 (PC61), CD122-PE-Dazzle594 (TM-β1), CD132-APC (TUGm2) (BioLegend). Samples were fixed and permeabilized with Foxp3 transcription factor staining buffer set according to manufacturer’s protocol (eBioscience) before staining for 45 minutes at 23°C with anti-mouse Foxp3-AF488 (FJK-16s, eBioscience) and Helios-Pacific Blue (22F6, BioLegend). Samples were washed once with stain buffer prior to data acquisition on a Cytek Aurora 5L (16UV-16V-14B-10YG-8R) spectral flow cytometer and analysis with FlowJo software (v10.6.1; Tree Star).