Caspase-3 Activity Protocol

A. Reagent Preparation

1. Reconstitute Ac-DEVD-AMC in 1 ml DMSO.
2. Thaw out reagents (DTT, Ac-DEVD-AMC) just before experiment.

NOTE: Precipitation may occur when reagents are stored at −20°C. Warm reagents to 37°C if necessary to dissolve precipitate.

3. Mix one part Assay buffer (2X) with one part dH₂O, and add DTT (1:200 dilution, final concentration of 5 mM) to make 1X assay buffer A.
4. Dilute Ac-DEVD-AMC (1:40 dilution) in 1X assay buffer A to make substrate solution B.

B. Cell Lysate Preparation: Collect lysate from 96-well plate

1. 1. Plate cells in 96-well plate and incubate with respective test substance for appropriate time. Typical cell count is 5x10⁴ – 2x10⁵ cells/well.
2. Following treatment, spin plate at 300xg for 10 min, remove the medium, rinse cells with ice-cold PBS, spin plate at 300xg for 10 min, remove PBS.
3. Add 30 μl/well of cell lysis buffer (#7018) and leave plate on ice for 5 min.

NOTE: Cell lysate plate can be stored at −80°C for future use.

Collect lysate from petri dish:

1. Check cell adhesion following treatment. If cells detach from the plate or are only loosely attached to plate, proceed to step b; if cells are tightly adhered to plate, proceed to step c.
2. Rinse plate with existing medium to collect all cells in a centrifuge tube. Spin at 1000xg cpm for 5 min, remove supernatant, and add cell lysis buffer (#7018) (0.5 ml/10 cm plate) to cell pellet. Pipette up and down a few times to break up the cells. Keep on ice and proceed to step d.
3. Rinse cells with ice-cold PBS, then add cell lysis buffer (#7018) (0.5 ml/10 cm plate) to plate and leave on ice for 5 min. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice and proceed to step d.
4. Sonicate lysates on ice.
5. Microcentrifuge for 10 min at 4°C and transfer the supernatant to a tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

C. Caspase Activity Assay

1. Dilute cell lysate in 1X assay buffer A to desired concentration (0.5–4 mg/ml is recommended). If cell lysates are from a 96-well plate, no dilution is necessary.
2. (Optional) Mix 25 µl of positive control AMC (supplied with kit) with 200 μl 1X assay buffer A to serve as a positive control.
3. Mix 200 µl of substrate solution B and 25 µl lysate solution in a black plate appropriate for fluorescent assay.

NOTE: We recommend reading the plate immediately and recording RFU reading at time 0 hr. This will help determine if there is significant change in RFU at the end of incubation.

NOTE: This protocol has been tested in 384-well plate format, please adjust the volume proportionally based on the plate capacity. For example, if using 384-low volume plate, use 20 µl substrate solution B and 2.5 µl lysate.

4. Incubate plates at 37°C in the dark.
5. Read RFU on a fluorescence plate reader with excitation at 380 nm and emission at 420–460 nm.

NOTE: We recommend reading plates after 1 hr incubation. If the signal is too weak, increase incubation period to observe significant change in signal strength. If significant increase is signal strength is not observed, more lysate may be necessary.