Reagents:
Iscove’s Modified Dulbecco’s Medium(IMDM) and HiSep were from HiMedia
Labs (Mumbai, India), Mesenchymal cell-specific fetal bovine serum
(Mesen FBS) and 35 mm2 low adhesion plates for colony
formation assays were from Stem Cell Tech (Vancouver, Canada);
Dynabeads® CD34 Positive Isolation Kit was from Invitrogen/Thermo Fisher
Scientific (MA); SCF, Granulocyte Monocyte Colony Stimulating factor
(GM-CSF) and Interleukin-3 (IL-3) were from Peprotech (NJ, USA),
Erythropoietin (EPO), TGFβ1, non-enzymatic cell dissociation solution,
Cyclopiazonic Acid (CPA), Indolactam (+), Indolactam (-), monensin,
antibody to cellular fibronectin (mouse monoclonal), were from
Sigma-Aldrich (MO, USA), BAPTA AM
[1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid
tetrakis(acetoxymethyl ester)], Thapsigargin, were from Molecular
Probes/Invitrogen/Thermo Fisher Scientific (MA,USA), cyclic RGD peptide
(AcPenRGDC-OH (Pen to C cyclised)), α4β1-specific peptide
(1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys-OH),
cell-permeant octapeptide PKC inhibitor (Myristoylated RKRTLRRL) and
Fibronectin-adhesion-promoting peptide were from Bachem AG, Bubendorf,
Switzerland, anti CD34 antibody (raised in mouse) was from BD
Biosciences (CA, USA), and anti-Ki67 (raised in rabbit) antibody was
from Abcam (Cambridge, UK) , FITC-conjugated anti-mouse and
PE-conjugated anti-rabbit antibodies were from Chemicon (CA, USA), all
tissue culture grade plastic ware was from Nunc/Thermo Fisher Scientific
( MA, USA) and microfuge tubes were from Eppendorf (Hamburg, Germany).
Dynabeads® CD34 Positive Isolation Kit was from Invitrogen/Thermo Fisher
Scientific (MA, USA).
Peptide sequences:
α5β1: H-*CRRETAWAC*-NH2 [ cyclised between C and C ]
αIIbβ3: H-*CNPRGD(109)RC*-NH2 [cyclised between C and C]
Most of the pharmacological reagents including all peptides were
dissolved in DMSO, unless otherwise stated. Control BMSCs were treated
with equal amounts of DMSO or its inactive control reagent dissolved in
DMSO. The working concentration of all these reagents was determined by
pre-screening them on BMSCs. The maximum tolerated non-toxic
concentrations were used in the experiments.