Cd105

Isolated hTGSCs, hDPSCs, and hPDLSCs (passage 3) were characterized for their mesenchymal cell surface profiles, as described previously.¹⁶ (link)^–18 (link) The hTGSCs and hDPSCs were trypsinized and incubated with the following conjugated antibodies: CD29, CD34, CD45, CD73, CD90, CD105, CD133, and CD166 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The hPDLSCs were then incubated with primary antibodies raised against STRO-1, CD146, CD90, CD44, CD19, or CD14. The cells were washed with phosphate-buffered saline (PBS) to remove the excess primary antibodies. The cells were analyzed by flow cytometry using a FACSCalibur flow cytometry system (Becton Dickinson, San Jose, CA, USA).

For FACS, cells were dissociated using 0.25% Trypsin/EDTA, washed with PBS, resuspended in blocking buffer consisting of 0.5% BSA and 2% FBS in PBS and kept for 30 min at 4 °C. Then, 1 × 10⁶ cells were incubated with antibodies against the human antigens CD34 (1:100, Santa Cruz Biotechnology), CD45 (1:100, Santa Cruz Biotechnology), CD73 (1:100, Santa Cruz Biotechnology), CD90 (1:100, Santa Cruz Biotechnology), and CD105 (1:100, Santa Cruz Biotechnology) for 1 h at 4 °C. A nonspecific isotype-matched antibody was used as a control. Afterwards, cells were incubated with a fluorescent dye conjugated secondary antibody (Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) (1:500, Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 546-conjugated rabbit anti-mouse IgG (H+L) (1:500, Invitrogen)) and diluted in FACS buffer (0.5% BSA and 0.05% sodium azide in 1× PBS) per the manufacturer’s recommendations in the dark for 30 min. After washing with FACS buffer, the cells were fixed using 4% paraformaldehyde buffer (Sigma, St. Louis, MI, USA) for 30 min and then analyzed using a flow cytometer (FACSCalibur Flow Cytometer, BD Biosciences).

We used flow cytometry to analyze cell surface
marker expressions by the ASCs culture
prior to ZnO-NPs treatment. The cells were
characterized according to a set of cell surface
markers characteristic for ASCs that included
cluster of differentiation (CD) CD73 (sc-14682,
Santa Cruz Biotechnology Inc., USA), CD105
(sc-20632, Santa Cruz Biotechnology Inc.,
USA), CD45 (sc-25590, Santa Cruz Biotechnology
Inc., USA) and CD34 (sc-7045, Santa
Cruz Biotechnology Inc., USA).The differentiation
potentials of the ASCs were checked in
specific media. For adipocyte differentiation,
cells were cultured in 1 μmol/l dexamethasone
(D4902, Sigma, USA), 60 μmol/l indomethacin
(I7378, Sigma, USA), and 450 μl 3-isobutyl-
1-methylxanthine (I5879, Sigma, USA). Adipocytes
were characterized by oil red-O (O0652,
Sigma, USA) staining under a light microscope.
For differentiation into osteoblasts, culture medium
was supplemented with 0.1 μmol/l dexamethasone,
10 mmol/l β-glycerophosphate
(G9422, Sigma, USA) and 60 mmol/l ascorbate
(A0157, Sigma, USA). Osteoblasts were characterized
by alizarin red (A5533, Sigma, USA)
staining and microscopic examination (16 (link)).

Adherent cells on passage 7 were resuspended in FACS (fluorescence-activated cell sorting) buffer, and the concentration adjusted to 10⁵cells/mL. For intracytoplasmic and nuclear markers, cells were permeabilized with 5 μl 0.1 % Triton X-100 for 30 min prior to incubation with primary antibodies (concentration of 1:100) specific for stem cells, inflammation, and cell cycle progression: Oct 3/4 (C-10, SC-5279), Nanog (n-17, SC-30331), CD45/OX1 (SC-53045), CD105 (2Q1707, SC-71042), CD90 (Thy-1, aTHy-1A1), CD34 (BI-3C5, SC: 19621), Caspase-3 (SC-7272), HSP-47 (SC-8352), P21 (SC-6246), Ki67 (Ab – 15580), Cyclin-D1 (AB-27618), P53 (Ab −26), TRA-1-81 (SC-21706), MCP-1 (SC- 32771), TNF-R1 (SC, 52746), all from Santa Cruz Biotechnology, as well as CD117 (c-Kit, SCF-Receptor Ab-6, RB-1518-R7, Thermo Scientific, Lab Vision Corporation, Fremont, CA, USA), VEGF-R1 (Clone VG1, M7273, DakoCytomation, CA, USA), COX-2 (Cayman Chemical Co, EUA), CD11b (MCA-551FT), Ly6a (Ab – 51317), CD1a (SC-18885), and CD133 (Mab4310, Merck Millipore), all for 45 min at room temperature. Then, cells were incubated for 2 h with a secondary antibody (Anti-Mouse FITC, DakoCytomation, Santa Cruz Biotechnology). The analysis was performed using a flow cytometer (FACSCalibur, BD). The expression of surface markers was determined by comparison with an isotype control analyzed by a histogram on the CELLQUEST program.

The fetal kidney cells were stained with following pre-conjugated or un-conjugated antibodies: CD44-fluorescein isothiocyanate (FITC) (BD Biosciences, CA, USA), CD90-FITC, CD45-phycoerythrin (PE), MHC class II-PE (all from Abcam, MA, USA) and with unconjugated CD29, VEGFR2, EpCAM (all from Abcam, MA, USA), CD73, CD24 (both from BD Biosciences, CA, USA), CD105 (Santa Cruz Biotechnology, Texas, USA), CD133 (My Biosource, CA, USA). After 30 minutes, cells incubated with un-conjugated antibodies were washed with PBS and stained with corresponding FITC-conjugated secondary antibodies. Cells stained with isotype-matched antibodies (IgG) served as controls. After staining, the cells were fixed in 4% para-formaldehyde (Sigma-Aldrich, MO, USA) and analyzed in FACS Calibur (BD Biosciences, CA, USA). The experiment was performed in three independent preparations.

Passage 2 cMSCs were analyzed with commercially available antibodies, acquired from AbD Serotec (CD9, CD34, CD44, CD45, CD90; Raleigh, NC, USA), Santa Cruz (CD105; Santa Cruz, CA, USA), and R&D Systems (STRO-1; Minneapolis, MN, USA) using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), CellQuest acquisition software (BD Biosciences), and FlowJo analysis software (TreeStar Inc., Ashland, OR, USA).