Specific for product: Caspase-3 Activity Assay Kit #5723.

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.

  1. 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.
  2. 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.

  1. Incubate plates at 37°C in the dark.
  2. 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.

posted October 2012

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