Rabbit cleaved caspase 3 antibody

HRMEC were cultured on gelatin‐coated coverslips placed in 24‐well‐ or 96‐well plates until confluence. On the next day, the medium was changed into plain EGM and cells were exposed to normoxia (21% O₂) or hypoxia (1% O₂) for 5 hours, followed by medium change to plain EGM with 100 ng/mL NGF or vehicle control and incubation for further 20 hours in normoxia or hypoxia. For the inhibition of the AKT pathway, Wortmannin (10 ng/mL; R&D Systems) was added in plain EGM 1 hour before the aforementioned 25 hour‐treatment. After washing, cells were fixed with 4% PFA for 15 minutes at room temperature followed by permeabilization and blocking in PBS including 5% goat serum and 0.3% Triton X‐100 for 2 hours and overnight incubation with rabbit cleaved caspase‐3 antibody (1:250; Cell Signaling Technology, Inc.) and FITC‐conjugated Isolectin GS IB4 (1:100; Life Technologies). After washings, cells were incubated for 1 hour with secondary Alexa Fluor 568‐conjugated anti‐rabbit antibody (1:350; Life Technologies) and DAPI (1:10 000) for nuclei visualization at room temperature. Random images were taken with an inverted fluorescence microscope (Zeiss, Oberkochen) (20×). More than 450 cells per condition were analysed in a blinded fashion with Fiji software. The percentage of cleaved caspase 3‐positive cells over the total cell number was calculated.

At day 7–10 of culture after reseeding dissociated neurospheres, differentiated cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. Cells were then blocked with 2% BSA in PBS for 30 min and stained with primary antibodies for 2 h at room temperature. The following antibodies were used: rabbit anti-TH antibody (PelFreez; 1:400), mouse anti-GFP antibody (MBL; 1:500), mouse anti-β3 tubulin antibody (Sigma-Aldrich; 1:400), mouse anti-Nestin antibody (Sigma-Aldrich; 1:200), and rabbit cleaved caspase-3 antibody (Cell Signaling Technology; 1:400). Cells were then washed three times and stained with Alexa Fluor 488- or 647-conjugated secondary antibodies (Thermo Fisher Scientific; 1:800) and DAPI (Thermo Fisher Scientific; 1:10,000) for 1 h at room temperature. Mitochondrial membrane potential and active mitochondrial mass for live cell imaging were detected by incubation for 30 min with 20 nM tetramethylrhodamine methyl ester (TMRM) (Thermo Fisher Scientific) and 100 nM Mitotracker Deep Red (Thermo Fisher Scientific), respectively. Immunostaining or live cell images were taken using a confocal microscope (Zeiss LSM880).

Apoptosis is a programmed cell death activated by a cascade of caspase enzymes. Expression of cleaved caspase-3 is a marker for the activation of the apoptotic signalling pathway. To measure cleaved caspase-3, Cos-7 cells treated with peptides for 48 h were fixed in 4% paraformaldehyde in PBS for 20 min. The cells were washed twice in PBS, permeabilized with 0.1% Triton X-100 for 5 min, blocked for 1 h with 1% BSA in PBS before adding rabbit cleaved caspase-3 antibody (Cell Signalling, Danvers, MA) (1: 400). The cells were washed with PBS and incubated with secondary antibody anti-rabbit IgG (1:300) + DAPI (1:300) for 1hr at RT covered from light. The cells were washed and observed under fluorescent microscope for cells expressing cleaved caspase-3.

THP1 cells (WT and SAM68 KO) were seeded onto glass coverslips placed on the bottom of a 12-well plate and treated with PMA for further differentiation, as described above. After 24-h incubation, cells were stimulated with cGAMP for 5 h or left untreated for control studies. Afterward, cells were treated for 40 min with 4% PFA following a 20-min permeabilization using 0.2% Triton X-100 in DPBS. Next, blocking with 2% FCS in DPBS was performed for 40 min, and rabbit cleaved caspase 3 antibody (1:400, Cell Signaling) or rabbit IFIT1 antibody (1:800, Cell Signaling) were applied for 1 h at room temperature. After three washes with DPBS, 5 mi each, the cells were incubated with goat anti-rabbit Alexa Fluor 488 nm fluorophore-conjugated secondary antibody (1:400, Invitrogen), Alexa Fluor Plus 647 Phalloidin (1:400, Invitrogen), and PureBlu DAPI Dye (1:100, Bio-Rad) for 1 h at room temperature in the dark. The cells were then washed three times with DPBS and mounted onto microscope slides using ProLong Gold Antifade mountant (Invitrogen). Slides were air-dried in the dark and examined on the next day using a Zeiss LSM 710 Inverted Confocal Microscope with corresponding Zeiss Zen software. Cleaved caspase 3-stained area was measured using ImageJ software.