Cd34 microbead kit ultrapure human

Citric acid (ACS reagent, ≥99.5%), Diethylentriamine (DETA, 99%), L-Glutamine-Penicillin-Streptomycin solution, Dulbecco’s Phosphate Buffered Saline (DPBS), Float-A-Lyzer dialysis devices (100–500 Da), human serum albumin, EDTA, Selenoprotein-AF647 antibodies and sterile filters (200 nm) were obtained from VWR and antibodies against CD45-FITC/CD34-PE, CD34-APC and the FITC Annexin V Apoptosis Detection Kit I were purchased from BD biosciences. Stem SPAN™ SFEM II medium, Stem SPAN™ CD34⁺ Expansion Supplement (10x) and Lymphoprep™ solution were bought at STEMCELL™ Technologies and microwave reaction vessels were obtained from CEM GmbH. The CD34 MicroBead Kit UltraPure human, MACS LS columns and 30 pre separation filters were purchased from Miltenyi Biotec and the Fix and Perm Kit was bought from Thermo Fisher Scientific. Separation buffer was prepared freshly by supplementing 500 ml DPBS with 1.5 ml 5% human serum albumin and 1.5 ml 50 mM EDTA.

esCAR T cells were purified using CD34 MicroBead kit UltraPure human (Miltenyi Biotec, cat# 130-100-453) with the MACS-LD column (Miltenyi Biotec, cat# 130-042-901) according to the manufacturer’s instructions. The extent of enrichment was checked by flow cytometry on a BD Accuri C6 flow cytometer.

Peripheral blood mononuclear cells (PBMCs) were separated by Histopaque (Histopaque 1077, Sigma-Aldrich) density gradient centrifugation from leukocyte-rich plasma named Buffy Coat, which was provided by the blood bank from healthy human donors. CD4 + CD25 + Treg cells, CD4 + Th cells, and CD8 + CTLs were isolated from PBMCs with magnetic bead-coupled antibodies (Miltenyi Biotec) and phenotyped with fluorochrome-coupled antibodies by flow cytometry as described (Supplement Fig. S1)^{59 (link)}. CD34-positive progenitor cells were isolated from PBMCs with the CD34 MicroBead Kit, UltraPure human (Miltenyi Biotec) (Supplement Fig. S2a and S2b).
PBMCs were cultivated in six-well Corning plates with RPMI and 1.5% autologous serum. Autologous serum was separated in the process of Ficoll (Histopaque 1077) density gradient centrifugation from buffy coat. After 30 min of cultivation (37 °C at 5% CO₂), monocytes attached to the bottom of the plates, whereas peripheral blood lymphocytes remained in the supernatant. The amount of CD3 + T cells within the PBLC population ranged between 70% and 85% (Supplement Figure S3a).
Jurkat cells (ACC 282, DSMZ) were cultivated in RPMI medium (Gibco Life Technologies) containing 10% fetal calf serum (Biochrome AG). Cells (2.5 × 10⁵) were irradiated and cultivated per well of a 12-well plate for cell death analysis.