Alexa fluor 700 mouse anti human ki 67 antibody

HaCaT cells were harvested and spun down to a pellet at 300 g for 5 minutes. The cells were washed with 1 mL of Phosphate Buffered Saline (PBS-1X) (Sigma-Aldrich) and centrifuged at 1500 rpm for 5 minutes. The cells were fixed with 4% p-formadehyde at room temperature for 30 minutes. Then, they were pelleted by centrifugation, resuspended with a permeabilization buffer (50 µL of buffer per 1 × 10⁶ cells). Afterwards, cells were centrifuged and stained with 5 µL of Alexa Fluor 700 mouse anti-human Ki-67 antibody (BD Pharmingen, #561277) and isotype control (BD Pharmingen, #557882) followed by room temperature incubation for 30 minutes protected from light. Finally, the cells were washed twice with 2 mL of 0.2% Saponin; resuspended and analyzed with a BD LSR Fortessa Flow Cytometer (BD Biosciences).

HaCaT cells were harvested and spun down to a pellet at 1500 rpm for 5 min. The cells were washed with 1 mL of phosphate buffered saline (PBS-1X) (Sigma-Aldrich) and centrifuged at 1500 rpm for 5 min. The cells were fixed with 4% p-formadehyde at room temperature for 30 min. Then, the cells were pelleted by centrifugation, resuspended with a permeabilization buffer (50 µL/mL PI, 100 µg/mL RNase, and 2 mM MgCl₂) (50 µL of buffer per 1 × 10⁶ cells). Afterwards, cells were centrifuged and stained with 5 µL of Alexa Fluor 700 mouse anti-human Ki-67 antibody (BD Pharmingen, #561277) and isotype control (BD Pharmingen, #557882), followed by room temperature incubation for 30 min, protected from light. Finally, the cells were washed twice with 2 mL of permeabilization buffer, resuspended, and analyzed with a BD LSR Fortessa Flow Cytometer (BD Biosciences).