MSC were cultured in MSC complete medium made up of Dulbecco’s Modified Eagle’s medium with nutrient mixture F-12 (HAM)[1:1] (DMEM/F12) with GLUTAMAX -I (Gibco, Invitrogen, USA), supplemented with 10% pre-selected foetal bovine serum (Stem Cell Technology Inc.), 1% of Penicillin /Streptomycin (Gibco, Invitrogen), 0.5% Fungizone (Gibco, Invitrogen), 0.1% Gentamicin (Gibco, Invitrogen), with or without 40ng/ml basic fibroblast growth factor (bFGF) (Peprotech, USA) . The serum used was pre-selected by the manufacturer to support MSC growth optimally in vitro culture while preserving MSC multi-potency to differentiate into chondro-, adipo- and osteogenic pathways.
3.3 T cell media
PBMC were cultured in complete T cell medium made up of RPMI 1640 (Gibco BRL, Invitrogen) supplemented with 10% foetal bovine serum (Gibco BRL, Invitrogen) or Human AB Serum and 1% Penicillin/Streptomycin (Gibco BRL, Invitrogen), 0.5% Fungizone (Gibco BRL, Invitrogen), 0.1% Gentamicin (Gibco BRL, Invitrogen).
3.4 Flowcytometry Analysis
The surface markers of cells were determined by direct immunofluorescence staining and analysed by flowcytometer. All antibodies were listed in Table 1. Cells were harvested and washed with 0.5% of BSA in 1X PBS (phosphate-buffered saline). Aliquots of cells from 105 to 106 were labelled with anti-human monoclonal antibodies, for 20 minutes at 2-8oC and washed with 0.5% of BSA in 1X PBS. All antibodies are raised in mice against human epitopes. The fluorochrome analysis wa...
... middle of paper ...
...idine (3H-TdR) incorporation assays
Proliferation assays were measured by tritium thymidine (3H-TdR) incorporation which reveals the proportion of cells in S phase of the cell cycle. In brief, at final 18 hours of incubation, 3H-TdR (0.037 MBq/well [0.5 μCi/well]) (Pelkin Elmer) were added to pulse the cultures. At the end of incubation, the cells were harvested onto glass fibre filter mats A (Perkin Elmer) using a 96 well plate manual cell harvester (MACH IIIM-FM, Tomtec, Inc. Hamden, CT USA). Scintillation cocktail was added to amplify the signal. Liquid scintillation spectroscopy was counted by Microbeta Trilux beta counter (Pelkin Elmer).
3.6 Statistical Analysis
Values for all measurements are presented as mean ± SD or stated otherwise. Comparisons for all pairs were performed by Student’s t-test. Significance levels were set at p value of 0.05.
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