Protocol for measuring the caspase-8 activity at the DED filaments in adherent cells

Figures

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  Figure 1. How to treat and harvest the cells (A) Picture of how to discard the media from adherent cells without stressing the cells or detach the cells from the plate. The tip of the aspiration pipette is in the edge of the plate. Lid is only slightly opened to ensure sterile conditions. (B) Picture of how to add fresh medium to adherent cells without stressing the cells or detach the cells from the plate. The tip of the pipette is in the edge of the plate. Lid is only slightly opened to ensure sterile conditions. (C) Before harvesting the cells place them on ice. (D) Scrap all the adherent cells from the plate with a cell scraper. Work on ice. (E and F) Transfer the cell suspension into a 50 mL tube. Work on ice.

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  Figure 2. How to wash and lyse the cells (A) Visible cell pellet after centrifuging the cells. Ensure that you see the pellet clearly. (B and C) Transfer the cells with PBS into a 1.5 mL tube. Ensure that you really transfer all of the cells. (D) Lyse the cells with 1 mL lysis buffer including 4% Protease Inhibitor Cocktail (PIC). Resuspend carefully until no clumps are visible. Place the tube on ice for 30 min.

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  Figure 3. Scheme of the method (A) Cells are seeded from a flask into three 14.5 cm plates per one condition. After the cells are harvested and washed, two samples are directly used for IPs. The third sample is separated in 100 μL for input control and 900 μL are used for IP. IPs from 1000 μL lysate are used for caspase-8 activity assay and IPs from 900 μL lysate are used for Western Blot control together with the input control. (B) An example of the Bradford Standard curve with measured values from the standard protein BSA. A linear regression was fit through the mean of triplicates. Calculating the protein concentration from this curve is only possible in the range between 0.5 and 5 μg/μL. The formula for calculating the protein concentration is shown in the corner as well as the coefficient of determination. Abbreviations: IP Immunoprecipitation.

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  Figure 4. CD95L induced caspase-8 activity at the DISC as measured by caspase activity assays (A and B) HeLa-CD95 cells were treated with CD95L for 2 h. Afterwards cells were harvested and DISC-IP was carried out using anti-APO1 antibodies. DISC-IP was analyzed using Western Blot with the corresponding antibodies. CD95 served as loading control for the IP (A) and actin served as loading control for the input (B). One representative Western Blot out of three is shown. (C and D) HeLa-CD95 cells were treated with CD95L for 2 h. Afterwards cells were harvested and DISC-IP was carried out using anti-APO1 antibodies. Caspase-8 activity at the DISC was measured using the Caspase-Glo 8 assay (Promega). All values are shown over the time (C) or two time points (120 and 180 min) were selected to show the bar diagram (D). Mean and standard deviation from three independent experiments are shown. Statistical analyzes were done with unpaired One-way ANOVA test with Tukey post hoc test. The following values were used: ∗∗∗∗p < 0.0001,∗∗∗p < 0,001; ∗∗p < 0,01; ∗p < 0,05; ns not significant Abbreviations: s.e. short exposure, l.e. long exposure, IP Immunoprecipitation, BC Beads control.