Moflo xdp cell sorter flow cytometer

For the proliferation assay, non adherent cells enriched for lymphocytes after monocyte depletion for DCs culture were prepared from allogenic and autologous PBMCs. Matured DCs primed with rhSPAG9 (250, 500, 750 and 1000 ng/ml per million cells), were co-cultured with PBMCs, stained with carboxyfluorescein, succinimidyl ester [CFSE (Invitrogen, Carlsbad, CA, USA)]. Briefly, SPDCs were co-cultured at the ratio of 1:50 (DCs: allogeneic PBMCs) and 1:10 (DCs: autologous PBMCs) to check for proliferation as described in our previous study [27 (link)]. The cells were cultured for 8 days. The cells were centrifuged at 1580 rpm and subsequently pellet was resuspended and washed with PBS, blocked with 5% FBS. The cells were further probed with anti-CD4-PC5, anti-CD8-APC, anti-CD56-PE, anti-CD25-ECD, anti-FOXP3-PE antibodies and Propidium iodide (1 µg/ml; Himedia, India) to verify the response of the autologous or allogeneic PBMCs upon stimulation with SPDCs. Unstimulated PBMCs were used as negative controls in all experiments and the percentage proliferation of these cells has been subtracted from all DC stimulated wells to arrive at the representative data. Cells were acquired using a MoFlo XDP cell sorter/ Flow cytometer (Beckman Coulter, Carlsbad, CA, USA) and FCS express 7 was used for analyzing the proliferating population.