Alexa fluor 488 mouse anti ki 67

The cultured cells were dissociated by TrypLE (Gibco) into a single cell suspension, and then fixed with 4 % paraformaldehyde and permeabilized by ice-cold methanol. Before staining, the sample cells were washed and resuspended at 5 ~ 10 × 10⁶ cells/ml in PBS containing 1 % BSA and 0.03 % NaN₃ (wash buffer) and then stained by the specific antibodies (PerCP-Cy™5.5 Mouse anti-Sox2, BD Biosciences; Alexa Fluor® 647 Mouse anti-GFAP, BD Biosciences; Alexa Fluor® 647 Mouse anti-Nestin, BD Biosciences; Alexa Fluor® 488 Mouse anti-Ki-67, BD Biosciences; PE anti-mouse CD133, Biolegend, San Diego, CA, USA). After antibody conjugate was added, the cells were incubated in the dark on ice for 30 min, and washed twice with wash buffer. The cell samples were resuspended and analyzed on a flow cytometer (Accuri C6, with the C6 software, BD Biosciences).

MRC5 cells were seeded in a 24 well plate until reach approximately a 10⁵ cells/mL density and then treated with 500ng/10⁵cells/mL of rhCCL11 for 2h or 24h. Cells were trypsinized and washed twice with FACS buffer (saline supplemented with 2% of FBS), fixed with BD Cytofix™ Fixation Buffer (BD Biosciences, San Jose, California) for 20 minutes at 4°C and permeabilized with BD Phosflow™ Perm Buffer III (BD Biosciences) for 30 minutes at 4°C. Cells were washed again with FACS Buffer and incubated with the following antibodies: Alexa Fluor® 647 Mouse anti-H2AX (pS139) 1:200 (#560447 BD Biosciences) and Alexa Fluor® 488 Mouse anti-p53 (pS37) 1:50 (#560282 BD Biosciences) for 30 minutes at 4°C on dark. For the proliferation assay cells were stained with Alexa Fluor® 488 Mouse anti-Ki-67 1:200 (#561165, BD Biosciences). Finally, cells were washed and resuspended in FACS buffer for acquisition (10.000 events).