Igg1k apc

Cells were washed twice with PBS and incubated in FACS-buffer (1% bovine serum albumin) with surface antibodies against CD71-VB405 (Miltenyi Biotec; 1:200), CD235-PE (Acris; 1:2500), CD14-APC (Miltenyi Biotec; 1:50), CD16-PE (BD Biosciences; 1:80); CD36-FITC (Pelicluster, Amsterdam, The Netherlands; 1:100); CD34-APC (IQ products, Groningen, the Netherlands; 1;10). Isotype controls were IgG1k-FITC (biolegends), IgG1-PE (Diaclone); IgG1k-APC (eBioscience). For hemoglobin staining on erythrocytes cells were fixed with 0.025% glutaraldehyde (sigma-Aldrich) and 0.5% paraformaldehyde (Signa-Aldrich) in PBS and permeabilized with 0.5% NP40 (Sigma-Aldrich) prior to staining HbA-PE (Santa Cruz; 1:1000) and HbF-APC (Invitrogen; 1:1000).

The immunophenotypic characterisation was performed using a fluorescence-activated cell sorting (FACS) analysis of cell-surface markers. Cells (passage 2) were labelled with monoclonal antibodies against CD31, CD34, CD45, CD73, CD90, CD106, CD146—FITC conjugate, CD105, CD29, CD44 and CD49c—APC conjugate (eBioscience, San Diego, CA, USA). Control samples were labelled with isotype-matched control antibodies IgG1K-FITC and IgG1K-APC (eBioscience). In brief, cells were trypsinised, aliquoted, fixed in 0.5% paraformaldehyde for 30 min at 4 °C and washed. Next, samples were incubated with either conjugated specific antibodies or isotype-matched control, diluted in 1× PBS supplemented with 5% FBS (FACS buffer). Labelled cells were washed, suspended in FACS buffer and analysed using a FC500 flow cytometer (Beckman Coulter, Lane Cove West NSW 2066, Australia).