Mouse anti cd44

1 × 10⁴ cell/cm² of hSCAPs were seeded on 24 well plate (Corning) and cultured for 2 days. The attached cells were fixed with 3.7% formaldehyde (Sigma) for 20 mins at room temperature, and fixed cells were washed with PBS. The cells were permeabilized with 0.2% Triton X-100 (Sigma) treatment for 20 mins, and, after brief washing with PBS, were treated with 4% bovine serum albumin (BSA: Sigma) to block non-specific binding of antibodies at 4°C for overnight. After blocking, the cells were incubated with primary antibodies such as 1:200-diluted mouse-anti-nestin (Abcam) antibody, 1:200-diluted mouse-anti-Stro-1 (Abcam) antibody, and 1:200-diluted mouse-anti-CD44 (Abcam), and 1:200-diluted mouse-anti-CD133 (Abcam) at 4°C for overnight. After reaction with primary antibodies, the cells were washed gently with PBS three times, and then incubated with secondary antibodies such as 1:2000-diluted alexa 488 (goat anti-mouse IgG, Invitrogen), 1:2000-diluted alexa 596 (goat anti-mouse IgG, Invitrogen) for 1 hr at room temperature under dark condition. Finally, after brief washing with PBS three times, the cells were counter-stained with DAPI, and mounted. The stained cells were observed under an inverted fluorescence microscope (Olympus, IX-72).

The slides of passage 3 BMSCs were fixed with 4% paraformaldehyde (Sinopharm, China) for 15 min and then rinsed 3 times using PBS. Afterwards, the cell slides were ventilated with 0.5% Triton X-100 (Beyotime, China) at room temperature for 20 min and blocked with goat serum for 30 min. A PBS wash followed each step. Diluted primary antibodies were added to the slides and coincubated at 4°C overnight. The primary antibodies used for immunostaining included mouse anti-CD90 (1/500, Abcam), mouse anti-CD11b (1/200, Abcam), mouse anti-CD44 (1/500, Abcam), mouse anti-CD29 (1/200, Abcam), rabbit anti-CD45 (1/100, Abcam), and rabbit anti-CD271 (1/500, Abcam). After 3 rinses with PBS, the slides were coincubated with diluted secondary primary antibodies (DyLight® 488-goat anti-rat IgG, 1/100, Invitrogen; DyLight® 488-goat anti-rabbit IgG, 1/100, Invitrogen) for 1 h. Following 3 times of PBS wash, the slides were coincubated with 4′,6-diamidino-2-phenylindole (DAPI) avoiding light for 5 min. After 4 subsequent PBS washes, the slides were dried using absorbent paper and mounted with an antifluorescence quenching agent (Southern Biotech, Alabama, US). Images were collected using a fluorescence microscope (Olympus BX53, Japan).

mesenchymal stem cells by flow cytometry
Pasage-3 adherent cells were treated with 0.25%
trypsin (Gibco, UK) and washed twice with PBS.
Cells were incubated with the antibodies mouse
anti-CD44, mouse anti-CD73, and mouse anti-
CD45 (Abcam, UK) for 30 minutes at 4˚C and resuspended
in 100 μl of PBS. (Gibco, UK) Unbound
antibodies were removed by washing with PBS. After
washing, the cells were incubated for 40 minutes at
room temperature in the dark using FITC conjugated
secondary antibody and re-suspended in PBS for
FACS analysis. At least 1×10⁶ cells per sample were
analyzed with a flow cytometer.