Anti active caspase 3

Expression of active caspase-3 and ZO-1 were analyzed using indirect intracellular flow cytometry. Briefly, HeLa, SiHa, and ME-180 cells were seeded in 6-well plates (3 X 10⁵ cells/well) in their respective culture medium and incubated overnight at 37 °C. The cells were then treated with MNBE for 24 h. Following incubation, cells were trypsinized, followed by washing in ice-cold PBS. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm Kit followed by incubation with anti-active-caspase-3 (1:250, BD Biosciences) and anti-ZO-1 (1:200, Invitrogen) antibody. Next, cells were washed twice and incubated in an APC-conjugated (Allophycocyanin) goat anti-rabbit antibody. Cells incubated with only APC-conjugated secondary antibody was used as a negative control. The stained cells were analyzed within one hour by flow cytometry (BD FACSVerse; BD Biosciences). Data were analyzed in FlowJo software. The experiments were performed in duplicates and repeated thrice.

The treated mice were euthanized on day 18 following tumor inoculation, and tumors were harvested. The tumors were dissected into fragments by cutting and digested by collagenase (0.5 mg/mL, MilliporeSigma) and DNase (1 μg/mL, MilliporeSigma) at 37°C for 45 minutes. The digested samples were then filtered through a 70 μm cell strainer and washed with PBS buffer. The cell pellets were incubated with RBC lysis buffer to lyse the RBCs. The cell suspensions were stained for the intracellular and extracellular protein markers of interest, and the stained samples were assessed on a flow cytometer (BD biosciences) along with CellQuest Pro software. The following staining antibodies used: anti-CD3, anti-CD4, anti-CD19, anti-Foxp3, anti-CD8, anti–granzyme B, anti–active caspase-3, and anti–IFN-γ (all from BD Biosciences).

Histological analysis was performed on snap frozen tissue. Tissue sections were fixed in acetone or 10% neutral buffered formalin, blocked with 5% FCS/0.3% Triton-X in PBS and stained with anti-active Caspase 3 (BD Biosciences), cleaved Caspase 8, cleaved Caspase-9, p-CREB (Ser 133), p-AMPK (Thr172) (all from Cell Signaling), CD8 and MHC-I (both from BD Biosciences) followed by incubation with the appropriate secondary antibodies. Cy3-conjugated anti-rabbit secondary antibodies were used for immunofluorescence. HRP-linked anti-rabbit secondary antibodies were used for conventional staining, which were visualised with the Peroxidase Substrate (ImmPACT NovaRED). Images were taken with an Axio Observer Z1 fluorescence microscope or Axiocam 503 color microscope (ZEISS) and quantified using Image J using the fluorescence intensity (MFI) per area as previously described [72 (link)].

Zebrafish protein was extracted from a pool of 15–20 de-yolked embryos and human protein was obtained from a confluent T75 flask of cultured fibroblasts, corresponding to approximately 10⁶ cells. The protein extraction, quantification, and Western blot was performed as previously described [46 (link)]. Ten micrograms of total protein were used. The total protein in the blot was quantified using stain-free gels and low-fluorescence PVDF membranes (BioRad, Hercules, CA, USA). Here, the trihalo compound in the stain-free gel binds the tryptophane in the proteins. Following activation with UV light, the total protein present in the gels and in the membranes becomes visible and the membranes are imaged for subsequent quantification. The antibodies used were rabbit anti-PDH-E1α pSer232 (AP1036, Merk), mouse anti-PDHA1 (45-6600, Invitrogen), anti-active caspase3 (559565, BD Biosciences, Stockholm, Sweden), goat anti-rabbit-HRP (A16110, Invitrogen), and goat anti-mouse-HRP (G-21040, Invitrogen). The signal detection was achieved using the Clarity of Clarity Max ECL Western Substrate (170-5060 and 170-562, BioRad) and the BioRad ChemiDoc MP imaging system.

For staining of active caspase-3, cells were washed with PBS, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin (Sigma-Aldrich). Cells were incubated with anti-active caspase-3 (BD Pharmingen, Heidelberg, Germany) in PBS/0.5% BSA/0.5% saponin for 20 min, stained with anti-rabbit-Alexa-Fluor647 (Dianova GmbH, Hamburg, Germany) for 20 min, and analyzed by flow cytometry.
AnnexinV-Propidium iodide staining was done by washing cells with annexin V-binding buffer (eBioscience) and staining with AnnexinV-FITC (1: 20; BD Pharmingen) 15 min at 4 °C. Propidium iodide (1 μg/ml; Sigma-Aldrich) was added and cells were analyzed on a FACS Calibur (Becton Dickinson, Heidelberg, Germany). The rate of dead cells was determined as a percentage of Annexin5/PI-positive cells (cells positive for annexin V or PI or both) of all cells analysed.
In some experiments, a fixable live-dead stain (LIVE/DEAD Fixable Far-Red Dead Cell Stain, Life Technologies, Carlsbad, USA) according to the manufacturer’s protocol was combined with staining for active caspase-3. After live-dead staining, samples were fixed in 4% paraformaldehyde for 30 min at RT, washed with PBS, and further subjected to the caspase-3 staining protocol as described above. Samples were analysed by flow cytometry on a FACS Calibur.

Embryos were fixed at the indicated stage in 4% PFA for 1–2 h at room temperature, permeabilized in PBST (PBS + 0.5% Triton X-100) and blocked in PBST + 2% bovine serum albumin at room temperature. Antibodies were diluted in PBST + 2% BSA and incubated overnight at 4°C or at room temperature for 4 h. Samples were washed in PBST for 2 h between antibody applications and cleared overnight in 70% glycerol. Primary antibodies used and their concentrations were as follows: antiphospho-Histone H3 (1:200, Abcam, ab14955); antiactive Caspase-3 (1:700, BD Pharmingen, 559565); anti-Pax2a (1:200, GeneTex, GTX128127); anti-F59 (1:10, DSHB); anti-Znp-1 (1:200, DSHB); anti-Zn-5 (1:200, DSHB); anti-SV-2 (1:200, DSHB); anti-GFP (1:200, Invitrogen, A10262); Alexa Fluor 568-conjugated Phalloidin (1:500, Invitrogen, A12380); and Alexa Fluor 488-conjugated α-bungarotoxin (10 µg/ml, Invitrogen, B13422). Secondary antibodies used were as follows: Alexa Fluor 488-conjugated goat antirabbit (Invitrogen, A11008); Alexa Fluor 488-conjugated goat antimouse (Invitrogen, A11001); Alexa Fluor 488-conjugated goat antichicken (Invitrogen, A11039); Alexa Fluor 568-conjugated goat antirabbit (Invitrogen, A11011); and Alexa Fluor 568-conjugated goat antimouse (Invitrogen, A11004), all used 1:200. TO-PRO-3 iodide (1 µM, Invitrogen, T3605) was used to detect nuclei.

FL-P or FL-D cells were plated in 24-well plates (10⁵ cells in 500 µl Hoxb8 progenitor or B cell medium) and treated with etoposide (Sigma Aldrich) or the inhibitors ABT-737, ABT-199, A-1155463 (Selleckchem) or S53845 (ApexBio) as indicated. For analysis of cell death induced by factor withdrawal, cells were washed and plated without FLT3L (FL-P-cells) or IL-7 (FL-D-cells). At various time points, cells were collected in PBS/4% FCS. Propidium iodide (Sigma Aldrich) was added immediately prior to analysis by flow cytometry (FACS-Calibur, Becton Dickinson). In some experiments, annexin V-Propidium iodide staining was used for quantification of apoptosis. Cell were washed with annexin V-binding buffer (eBioscience) and stained with annexin V-FITC (1:20, BD Pharmingen) and PI (5 µg/ml) for 20 min at 4 °C followed by flow cytometry analysis (FACS Calibur). For staining of active caspase-3, cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% saponin (Sigma-Aldrich). Cells were incubated with anti-active caspase-3 (BD Pharmingen) in PBS/0.5%BSA/0.5% saponin for 30 min, stained with anti-rabbit-Alexa-Fluor488 (Dianova GmbH, Hamburg, Germany) for 30 min and analyzed by flow cytometry (FACS Calibur). Routinely, three biological replicates were performed. Further replicates were done depending on the statistical distribution of the values.