Caspases,Paracaspases, and Metacaspases Methods and Protocols

167

All incubations should be carried out at room temperature unless

otherwise noted, in a humid covered dish or plate to prevent dry-

ing and fl uorochrome fading.

1. Aspirate PBS and incubate cells in blocking/permeabilizing
   buffer for 60 min.


2. While blocking, dilute the primary antibody of interest in anti-
   body dilution buffer ( see Note 34 ).


3. Using a thin forceps, pick up the coverslips and put them cell
   side up in a humid light-tight plate. Apply 70 μL of diluted
   primary antibody to each coverslip ( see Note 35 ).


4. Incubate overnight at 4 °C.


5. Aspirate the primary antibody solution and rinse two to three
   times in 70–100 μL PBS for 5 min each.


6. Apply 70 μL fl uorochrome-conjugated secondary antibody
   (usually 1:200) diluted in PBS and incubate for 1–2 h at room
   temperature in dark.


7. Wash specimens as in step 5.


8. Counterstain the nuclei for 10–15 min with 70 μL DAPI or
   Hoechst 33342 (both 1 μg/mL in PBS).


9. Wash once in PBS for 5–10 min.


10. Mount coverslips on specimen glass plates using 2–3 μL
    VECTASHIELD or equivalent and seal with nail polish.


11. For best results, allow mountant to cure overnight at room
    temperature. For long-term storage, store slides fl at at 4 °C
    protected from light.


12. Analyze the slides in a confocal microscope.

The method includes the same steps as described in Subheading 3.1.

1. Induce apoptosis in cells by desired method. Concurrently
   incubate a control culture without induction.


2. Harvest and collect 1 × 10^6 cells in a tube. Centrifuge for 5 min
   at 150 × g , 4 °C.


3. Wash cells in PBS, centrifuge again, and discard supernatant.


4. Repeat step 3.


5. Fix the cells by resuspending in 0.5 mL ice-cold pure methanol
   at −20 °C for 30 min ( see Notes 36 and 37 ).


6. Wash the fi xed cells twice in PBS.


7. Dilute the M30 Cytodeath-FITC antibody to 1 μg/mL in
   dilution buffer and incubate the cells with 100 μL of the work-
   ing solution for 30–60 min at room temperature in the dark.

3.2.2 Immunostaining

3.3 Immunoblot
Analysis of Caspase
Substrate Cleavage

3.4 Detection of
Cytokeratin 18
Cleavage by Flow
Cytometry

3.4.1 Sample
Preparation

3.4.2 Detection of the CK
18 Neoepitope

Caspases in Mammalian Cell Cultures