Sepmate 50 separation tubes

Experimental protocols were approved by the USC Institutional Review Board (IRB) and conducted in accordance with the principles of the Declaration of Helsinki. Human blood ILC2s were isolated from total peripheral blood mononuclear cells (PBMCs) to a purity of >95% on a FACSARIA III system. A total of 14 healthy volunteers (7 males, 7 females) with written consent participated in the study, aged 18–70, no compensation was provided. Briefly, human fresh blood was first diluted 1:1 in PBS 1X and transferred to SepMateTM-50 separation tubes (STEMCELL Technologies) filled with 12 mL Lymphoprep™. Samples were centrifuged for 10 min and PBMCs were collected. CRTH2⁺ cells were then isolated using the CRTH2 MicroBead Kit, used according to the manufacturer’s conditions. Samples were then stained and ILC2s were isolated based on the absence of common lineage markers (CD3, CD5, CD14, CD16, CD19, CD20, CD56, CD235a, CD1a, CD123), and the expression of CD45, CRTH2 and CD127. Isolated ILC2s were cultured at 37 °C (5 × 10⁴/mL) with recombinant human (rh)IL-2 (10 ng/mL) and rhIL-7 (10 ng/mL) in cRPMi.

For human ILC2s from blood, peripheral blood mononuclear cells (PBMCs) were first isolated from fresh human blood by diluting the blood 1:1 in PBS and adding to SepMate^TM-50 separation tubes (STEMCELL Technologies Inc, Vancouver, Canada) prefilled with 15 mL Lymphoprep^TM each (Axis-Shield, Oslo, Norway) and centrifugation at 1200 × g for 15 mins. Human ILC2s were then isolated by cell sorting based on the lack of expression of classical lineage markers (CD3, CD5, CD14, CD16, CD19, CD20, CD56, CD235a, CD1a, CD123) and expression of CD45, CRTH2 and CD127. Purified human ILC2s were stimulated (5 × 10⁴ per mL) with recombinant human (rh)-IL-2 (20 ng/mL), rh-IL-7 (20 ng/mL) for the indicated times at 37 °C in presence or absence of rh-IL-33 (20 ng/mL) and/or TL1A-L (Fc-Fusion) from AB Bioscience (P7042F).

Experimental protocols were approved by the USC Institutional Review Board (IRB) and conducted in accordance with the principles of the Declaration of Helsinki. Human blood ILC2s were isolated from total peripheral blood mononuclear cells (PBMCs) to a purity of > 95% on a FACSARIA III system as described previously (29 (link)). Briefly, human fresh blood was first diluted 1:1 in PBS 1X and transferred to SepMateTM-50 separation tubes (STEMCELL Technologies) filled with 12mL Lymphoprep™. Samples were centrifuged for 10 minutes and PBMCs were collected. CRTH2⁺ cells were then isolated using the CRTH2 MicroBead Kit, used according to the manufacturer’s conditions. Samples were then stained and ILC2s were isolated based on the absence of common lineage markers (CD3, CD5, CD14, CD16, CD19, CD20, CD56, CD235a, CD1a, CD123), and the expression of CD45, CRTH2 and CD127. Isolated ILC2s were cultured at 37°C (5x10⁴/mL) with rhIL-2 (10ng/mL) and rhIL-7 (10ng/mL) in complete RPMI (cRPMI). To make cRPMI, RPMI (Gibco) was supplemented with 10% heat-inactivated FBS (Omega Scientific), 100 units/mL penicillin and 100mg/mL streptomycin (GenClone). When indicated, human ILC2s were activated with 50ng/mL rhIL-33 for the indicated times. Human PD-1 blocking antibody or isotype was included in the culture (10µg/ml, BioXCell).