Anti ki67 clone sola15

Single cell suspensions (1 × 10⁶ cells/100 μl) were prepared with dead cells excluded by using Zombie Aqua Fixable Viability Kit (Biolegend, USA). After being washed, cells were resuspended in buffer (PBS containing 5% FBS) and were blocked in Fc block (Anti-mouse CD16/32, Clone 2.4G2, BD, USA) at 4°C for 30 min. Cells were then incubated with following various antibodies for 30 min at 4°C. anti-CD11b (clone M1/70, Biolegend, USA), anti-F4/80 (clone BM8, Biolegend, USA), anti-Ly-6G/Ly-6C (clone RB6-8C5, eBioscience, USA), anti-CD11c (clone N418, Biolegend, USA), anti-I-A/I-E (clone M5/114.15.2, Biolegend, USA); Rat IgG2b,ҝ Isotype Ctrl (Biolegend, USA); anti-CD3 (clone 17A2, Biolegend, USA), Anti-CD4 (clone RM4-5, BD, USA), anti-CD8α (clone 53–6.7, BD, USA), anti-CD44 (clone IM7, BD, USA); anti-CD62L (clone MEL-14, eBioscience, USA), anti-Ki67 (clone SolA15, eBioscience, USA). Ki67 staining was implemented using Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) according to manufacturers’ instructions.

The mammary tumors were dissected from the mice at the indicated time points. The tissues were cut into pieces and digested with collagen IV (1 mg/ml, sigma) and Dispase II (1 mg/ml, Sigma) and they were shaking for 1 h at 37 °C. The tissue/media mixture was strained using a 70 μm cell strainer for single cell suspension preparation. The cells were added anti-mouse CD16/CD32 (Clone 93, Biolegend) and incubated on ice for 10 min. Thereafter they were labeled with anti-mouse PDGFRα (Clone APA5, Biolegend) and anti-mouse F4/80 antibodies (Clone BM8, Biolegend) at a 1:100 dilution for 30 min on ice. Cells were fixed and permeabilized using Fixation/permeabilization concentrate (Ebioscience) and labeled with anti-Ki67 (Clone SolA15, Ebioscience). The FACS data were acquired using a CantoII flowcytometer (BD) and analyzed with FlowJo software. The living PDGFRα⁺F4/80⁻ CAFs were sorted using Aria II cell sorter (BD Bioscience).
Sorted CAFs were seeded in 24-well plates in DMEM containing 10% FBS and 100 units/ml penicillin/streptomycin. Subsequently, non-adherent cells were removed by extensive washing with PBS and adherent cells were treated as indicated for further analysis.