All cell culture procedures were performed under sterile conditions in laminar class II biohazard safety cabinet (ESCO). The cell cultures were incubated at 37oC in 5% CO2 humidified incubators (RSBiotech).
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 was included with appropriate FITC-, PE-, PE-CY5-, PERCP-, APC-, conjugated isotype controls. The stained samples were assessed by using the FACSCalibur flowcytometer (Becton Dickinson). Gating at FACS acquisition was applied to select the target cells population and exclude any dead cells and debris. Ten to hundred thousands of events/cells were acquired and the data were analysed using CellQuestPro software by manufacturer.
Antibody Predominant reactivity Clone/source
Anti-huCD105-PE Endoglin 166707/R&D System
Anti-huCD73-PE T, B, DC & stem cells AD2/BD
Anti-huCD90-FITC Thy-1 cells 5E10/BD
Anti-huCD29-APC thrombocytes, monocytes, MAR4/BD
Anti-huCD45-PerCP leucocytes 2D1/BD
Anti-huCD34-FITC HSC 8G12/BD
Anti-huCD80-FITC Activated B cells & DC L307.4/BD
Anti-huCD86-FITC Activated B cells & monocytes 2331(FUN-1)/BD
HLA-A,B,C-PE-Cy5 MHC-I expressing cells G46-2.6/BD
HLA-DR,DP,DQ-FITC MHC-II expressing cells TÜ39/BD
Anti-huCD4-PerCP CD4 T cells SK3/BD
Anti-huCD8-APC CD8 T cells SK1/BD
Anti-huCD25-FITC Activated T cells & B cells 2A3/BD
Anti-huCD69-PE Activated T, B & NK cells L78/BD
Anti-huLAP(TGF-β1) cells expressing LAP (TGF-β1) 27232
FITC Fluorescein isothiocynate
PERCP Peridinin chlorphyll protein
DC Dendritic cells
NK Natural Killer
Table 1: Anti-human antibodies used for flowcytometric analysis. All antibodies were purchased from Becton Dickinson/Pharmingen except CD105 and LAP (TGF Beta I) from R&D System. All antibodies were used at volume 10µl/106 cells in 100µl of total staining volume.
3.4.2 Cell Cycle Analysis
DNA content was identified using propidium iodine (PI). Briefly, cells were harvested from experimental culture, washed and then fixed in 70% alcohol in -20oC for overnight. Then, the fixed cells were washed and incubated with 100µg/ml Propidium Iodide (PI) (Molecular Probe, Invitrogen) and 20ng/ml RNase (Sigma) in PBS for 30 minutes. In T cells cell cycle analysis (section 6.2.5), 100ng/ml of ﬂuorescein isothiocyanate (FITC) was added into the staining buffer to identify the intracellular protein. The results were acquired by flowcytometry and analysis was done by FCS Express V3.
3.5 Tritium thymidine (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.