Anti cd45ra apc h7

Anti-CD127-PECy7, anti-CD25-PECy5, anti-CD45RA-APCH7, anti-CD19-FITC, and anti-CD3-APCCy7 were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD4-PE and anti-CD8-PECy7 were purchased from Tonbo Biosciences (San Diego, CA, USA). Anti-FOXP3-AlexaFluor647 was purchased from Beckman Coulter (Brea, CA, USA). Carboxy fluorescein succinimidyl ester (CFSE) was purchased from Life Technologies (Eugene, OR, USA).
For intracellular staining, FOXP3/Transcription Factor Staining Buffer Set (eBiosciences, San Diego, CA, USA) was used. All staining were performed in RPMI 1640 (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS), 2 mM glutamine, 10 mM HEPES, and 10 mM antibiotic-anti-mycotic (Gibco).

Umbilical cord blood was collected in several hospitals using Stemcare/CB collect blood bag system (Fresenius Kabi Norge AS) containing citrate phosphate dextrose (CPD) as an anticoagulant. Approval for collection was obtained from the Medical Ethical Committee of the Erasmus University Medical Centre (MEC-2009–410) and written informed consent from the mother was obtained prior to donation of the cord blood. Within 48 hours after collection, mononuclear cells were isolated using ficoll (Lymphoprep, Fresenius Kabi Norge AS). CD34⁺ cells were isolated with double positive immunomagnetic selection using Magnetic Activated Cell Sorting (MACS) technology according instructions of the manufacturer (Miltenyi Biotech GmBH, Bergisch Gladbach, Germany). MACS-selected CD34⁺ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI^-Lin^-CD34⁺CD38^lowCD45RA^lowCD90⁺-cells, highly enriched for hematopoietic stem cells (HSC)[30 (link)], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA).

In total, 0.5–1 × 10⁶ cells were harvested from the 16-h or 10-day stimulation cultures, washed, and incubated with Live/Dead Aqua V510 for 15 min on ice. The cells were then washed again and surface-stained for 30 min on ice with the following antibodies: anti-CD3-FITC (BioLegend, clone UCHT1, Cat# 561802), anti-CD4-BV421 (BioLegend, clone OKT4, Cat# 317434), anti-CD8-PerCPCy5.5 (BD Pharmingen™, clone RPA-T8, Cat# 560662), anti-CD27-PE (BD Pharmingen™, clone M-T271, Cat# 555441), and anti-CD45RA-APC-H7 (BD Pharmingen™, clone HI100, Cat# 560674). After fixation and permeabilization with Cytofix and Perm (BD Bioscience, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min with anti-TNF-PE-Cy7 (BD, clone MAb11, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, Cat# 554702). After the final wash, the cells were resuspended in 200 μL FACS buffer. A FACSFortessa instrument (BD Bioscience) was used to acquire data, which were analyzed using FlowJo software (Treestar).