Fig. 2: Ferroptosis induction leads to phosphatidylserine exposure and can be detected with commercially available dyes AnnexinVivo and CJ215.

a An Annexin V staining of cells treated with erastin (15 μM) for 6 and 18 h and RSL3 (1 μM) treated for 2 h either with and without Liproxstatin (2 μM) shows an increased fluorescence upon ferroptosis induction which can be decreased with liproxstatin. 18 h staurosporine treatment is used as a positive control. b Measuring the fluorescence levels in MDA MB 435 cells treated with erastin, RSL3 and staurosporine using three different dyes AnnexinVivo, ICG and CJ215 showing the effectiveness of CJ215 over the other contrast agents. c Measurement of fluorescence upon 24 h of erastin treatment (10 μM) along with ferroptosis inhibitors using AnnexinVivo staining. d Measurement of fluorescence upon 24 h of erastin treatment (10 μM) along with ferroptosis inhibitors using CJ215 staining. e Measurement of fluorescence upon 6 h of RSL3 treatment (1 μM) along with ferroptosis inhibitors using CJ215 staining. f Representative image of erastin-treated cells with and without ferroptosis inhibitors using CJ215 staining. g Representative image of RSL3-treated cells with and without ferroptosis inhibitors using CJ215 staining. h Fluorescence intensity in CJ215-stained cells treated with erastin and staurosporine with or without addition of Annexin V proteins show decreased fluorescence upon Annexin V blocking. N = 5 pooled from three independent experiments. Mean ± s.d. p values are reported above the lines (Ordinary one-way ANOVA with Tukey’s multiple comparisons).