Cd34 apc

Cell preparation and staining was performed as previously described with minor modifications^{5 (link)}. Mononuclear cells (MNCs) were purified by Histopaque 1077 (Sigma-Aldrich) gradient centrifugation. The MNC fraction was washed by centrifugation and resuspension twice in HBSS at room temperature to remove residual platelets. The cell concentration in each sample was then enumerated using an automated cell counter (Beckman Coulter AcT diff hematology analyzer). Five million MNCs were stained for 20 minutes with a cocktail containing saturating amounts of the following mAbs: CD45RA-FITC (Clone ALB11; Beckman Coulter), CD123-PE-Cy7 (Clone 9F5; BD Biosciences), CD38-PerCP-Cy5.5 (Clone HIT2; BD Biosciences), CD34-APC (Clone BIRMA-K3; Dako), CD90-BV421 (Clone 5E10; BD Biosciences), and CD133−PE (Clone AC133; Miltenyi Biotec). Live Dead Aqua (Thermo Fisher) was used to enumerate and exclude dead cells from the analysis. After incubation with mAbs the cells were washed once with PBS containing 0.5% bovine serum albumin, 0.1% sodium azide and 0.0004% tetrasodium EDTA.

Mononuclear cells (MNCs) were purified by Histopaque 1077 (Sigma-Aldrich) gradient centrifugation. The MNC fraction was washed twice by centrifugation and resuspended in HBSS at room temperature to remove residual platelets. The cell concentration in each sample was then enumerated using an automated cell counter (Beckman Coulter AcT diff hematology analyzer). Five million MNCs were stained for 20 minutes with a cocktail containing saturating amounts of the following mAbs: CD45RA-FITC (Clone ALB11; Beckman Coulter), CD123-PE (Clone 9F5; BD Biosciences), CD38-PerCP-Cy5.5 (Clone HIT2; BD Biosciences), CD127-PerCP-Cy-7 (Clone HIL-7R-M21; BD Biosciences), CD34-APC (Clone BIRMA-K3; Dako), CD90-BV421 (Clone 5E10). Live Dead Aqua 405 nm (Invitrogen) was used to enumerate and exclude dead cells from the analysis. After incubation with mAbs the cells were washed once with PBS containing 0.5% bovine serum albumin, 0.1% sodium azide and 0.0004% tetrasodium EDTA. We compared the effect of sample preparation on the quantity of CD34+ cell populations obtained using whole blood staining followed by ammonium chloride based RBC lysis versus Ficoll density gradient separation of mononuclear cells. We found negligible differences between the two sample preparation methods on the quantity and percentages of CD34+ cell populations obtained (Supplemental Table 1).