Guava easycyte

After cell harvesting and fixation, fluorescein isothiocyanate (FITC) conjugated 8-oxodG antibody (1:10,000 dilution) (Santa Cruz Biotechnology) were chosen for flow cytometry reaction at 4 °C for 1 h and analyzed by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson).

MitoMP-sensitive probe DiOC₂(3) (Invitrogen, Eugene, OR, USA) (5 nM, 37 °C, 30 min) was used to determine MitoMP by a flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) [31 (link)].

1 × 10⁵ cells were seeded in 12-wells plates and treated with TMZ (100 μM), Erastin, RSL3 (1 μM), and Ferrostatin-1 (1 μM) for 24 h. Briefly, cells were collected, washed with PBS and re-suspended in 10 μM DCFDA (2′,7′-dichlorofluorescein diacetate, Invitrogen) or 2 μM BODIPY C11 (581/591) (Invitrogen) probe diluted in PBS. After 30 min of incubation, analysis was performed with Guava EasyCyte or BD LSRFortessa flow cytometers, and analyses were carried out with FlowJo software (BD Biosciences). For microscopic analysis, 1 × 10⁴ cells were grown in six-well plates and stained with DCFDA or BODIPY-C11 for 30 min, then the pictures were taken by Inverted Microscope Axio Observer Z1 Zeiss. All imaging acquisition parameters were kept constant for each sample.

Flow cytometry was used to quantify the expression levels of BFP. The BFP-HEK and BFP-K562 cells were analyzed 5 days after Cas9 treatment. The BFP-HEK cells were washed with PBS and detached by trypsin. BFP and GFP expression were quantified using a BD LSR Fortessa X-20 and Guava easyCyte.