For the rapid, sensitive and accurate measurement of apoptosis in various samples.

Transcription

1 ab Caspase Cascade Screening Kit Instructions for Use For the rapid, sensitive and accurate measurement of apoptosis in various samples. This product is for research use only and is not intended for diagnostic use. Version 2 Last updated 16 April 2015

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4 Table of Contents 1. Overview 2 2. Protocol Summary 3 3. Components and Storage 4 4. Assay Protocol 5 5. Troubleshooting 6 3

5 1. Overview Activation of members of caspase-family proteases plays a key role in apoptosis. Abcam s Caspase Cascade Screening Kit provides a convenient means for detecting activation of caspases by flow cytometry in living cells. The assay is based on the cleavage of (aspartyl) 2-Rhodamine 110 (D 2 R), a reported substrate for members of caspase family proteases. The caspase substrate D 2 R is non-fluorescent, however, upon cleavage of the substrate by cellular caspases, the released rhodamine 110 gives rise to fluorescence that can be measured at excitation of 488 nm and emission of 530 nm. 4

6 2. Protocol Summary Induce Apoptosis in Sample Cells Add D 2 R Incubation Buffer Add DTT Add D 2 R Reagent and Incubate Quantify Using Flow Cytometry 5

7 3. Components and Storage A. Kit Components Item Quantity D 2 R Reagent DTT (1 M) D 2 R Incubation Buffer 100 μl 300 μl 30 ml Store at -20 C. After thawing, store the Incubation Buffer at +4 C. Protect D 2 R reagent from light. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Flow Cytometer or Fluorescence Microscope Glass slides and coverslips 6

8 4. Assay Protocol a) Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. b) Count cells and pellet 1 x 10 5 cells by centrifugation. c) Resuspend cells in 300 μl of D 2 R Incubation Buffer d) Add 3 μl of the 1 M DTT (10 mm final concentration). e) Add 1 μl of D 2 R Reagent f) Incubate at 37C for min in the dark g) Analyze cells by flow cytometry using the FL-1 channel (Ex/Em = 488/530 nm) Note: This product detects proteolytic activity. Do not use protease inhibitors in the sample preparation step as it might interfere with the assay. For further technical questions please do not hesitate to contact us by (technical@abcam.com) or phone (select contact us on for the phone number for your region). 7

9 5. Troubleshooting Problem Reason Solution High Background Cell density is higher recommended than Increased volumes of components added Incubation of cell samples for extended periods Refer to datasheet and use the suggested cell number Use calibrated pipettes accurately Refer to datasheets and incubate for exact times Use of extremely confluent cells Perform assay when cells are at 80-95% confluency Contaminated cells Check for bacteria/ yeast/ mycoplasma contamination 8

10 Problem Reason Solution Lower signal levels Cells did not initiate apoptosis Very few cells used for analysis Incorrect setting of the equipment used to read samples Use of expired kit or improperly stored reagents Determine the time-point for initiation of apoptosis after induction (time-course experiment) Refer to data sheet for appropriate cell number Refer to datasheet and use the recommended filter setting Always check the expiry date and store the components appropriately Erratic results Uneven number of cells seeded in the wells Adherent cells dislodged at the time of experiment Incorrect incubation times or temperatures Seed only healthy cells (correct passage number) Perform experiment gently and in duplicates or triplicates for each treatment Refer to datasheet & verify correct incubation times and temperatures Incorrect volumes used Use calibrated pipettes and aliquot correctly 9

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13 UK, EU and ROW Tel: +44- (0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Version 2 Last updated 16 April 2015 Copyright 2015 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.