Pe mouse anti human ki67 antibody

HEKa and M249 cells treated with a concentration of 1.5 μM of vemurafenib or vehicle control were fixed with formaldehyde to a final concentration of 1.6%, and permeabilized using methanol (90%). Then, they were washed in staining buffer (sterile PBS, 0.5% BSA, 0.01% sodium azide, NaN₃), and stained with Alexa Flour 647 mouse anti-ERK1/2 (pT202/pY204) antibody and PE mouse anti-human Ki67 antibody (BD Pharmingen, San Jose, CA, USA). After incubation, cells were washed again and resuspended in 3 ml of staining buffer. A total of 30,000 cellular events were acquired for analysis. Data was analysed using FlowJo (Tree Star Inc., Ashland, OR, USA). HEKa and M249 cells were routinely tested for mycoplasma and were found to be negative.

TM00386 PDXs established in ovariectomized SCID/beige mice were harvested and dissociated using digestion buffer [125 μg/mL DnaseI (#LS002138, Worthington), 10 μg/mL Dispase (#LS02109, Worthington), 500 μg/mL Collagenase 3 (#LS004182, Worthington), and 5× triple antibiotics (#15240-062, Invitrogen)] to generate single cells. The cells (~2 × 10⁶) were washed with PBS and fixed in 99% methanol for 3 h at −20 °C. Cells were then incubated with 20 μL of PE Mouse anti-Human Ki67 antibody (#51-36525, BD Pharmingen) or 20 μL of PE Mouse IgG1 (#51-35405, BD Pharmingen). FACS analysis was performed on the LSRFortessa X-20 Cell Analyzer (BD Biosciences), and the data were analyzed with FlowJo software. The remaining half of the PDXs was fixed in 10% neutral buffered formalin followed by embedding in paraffin for Ki67 IHC.