FAM-FLICA Caspase 3 and 7, assay kit
Full name of the product
FAM-FLICA Caspase 3 and 7 Assay Kit
Product Gene Name
FAM-FLICA Assay Kit
The FLICA (Fluorescently Labeled Caspase Inhibitor) Caspase 3 and 7 Assay is a simple but accurate assay to measure apoptosis via caspase 3 activity in whole living cells. Four example protocols are described below.
Culture your cells to 1 x 10^6 cells/mL. Follow the experimental protocol where caspase activity will be investigated; Create positive and negative controls for caspase activity. Reconstitute reagent with 50 ul DMSO to form stock concentrate (can be frozen for future use). Dilute the stock concentrate with 200 ul of 1X PBS to form the working solution.
Add ~10 ul of the working solution directly to a 300-500 ul aliquot of your cell culture to be labelled. Incubate 30 minutes -1 hour. Wash and centrifuge cells two to three times, or allow to incubate for 1 hour with fresh medium or 1x apoptosis wash buffer. If desired, label cells with Hoechst stain. If desired, label cells with propidium iodide or 7-AAD. If desired, arrange the cells. Analyze the data with a fluorescence microscope, plate reader, or flow cytometer.
Prepare frozen tissues according to the experiment. Allow the slides to air dry. Fix the slides with acetone for 1 minute. Rehydrate slides by washing (twice for 5 min) in TBS-tween (TBSt) or PBS-tween (PBS). Block slides for 20 min (as 20% Aquablock in media with 0.2% interpolation). Dilute 150X FLICA stock 1:50 in PBS to form a 3X working solution.
For example, add 50 uL of 150X stock to 2450 uL of PBS (2.5 mL total). Add 50 uL of 3X FLICA and incubate for>1hr protected from light. Wash with TBSt or PBS (twice for 5 min) by placing the slides in a slide incubator plate containing 1X Wash Buffer. Develop with DAPI and coverslips. Store samples at 2-8 degrees C for short-term storage, staining will last at -20 degrees C for extended periods.
Adherent cells must be washed carefully to avoid the loss of any cells that round off and detach from the plate surface. Loose cells can be collected from the surface of the plate or slide and treated as cells in suspension, while those remaining attached to the surface should be washed as attached cells. If the adherent cells are trypsinized, the loose cells can be recombined with the trypsinized pool, or the washed loose cells can be recombined with the adherent portion when performing analysis.
If adherent cells are grown on a tissue culture plate, the entire plate can be gently rotated as part of the washing process to pellet loose floating cells. Avoid any attempt to trypsinize the cells before labelling them with a vital stain such as PI. Cell membranes exposed to trypsin could become transiently permeable to vital dyes for a variable period of time, depending on the cell line. Cells can be labelled with FLICA before or after trypsinization.
Adherent cells: trypsinization before FLICA labelling and FACS analysis
Culture cells in T25 flasks and exposure to experimental conditions. Apoptotic cells can detach and begin to float in the medium. Save and centrifuge to pellet and include these cells in your analysis. Trypsinize adherent cells; neutralize with trypsin inhibitor present in 20% FBS cell culture medium; group the cells with the granules created in #2; add a few ml of media.
Centrifuge ~5 min at 220 x g and remove all but ~100 uL of supernatant. Count the cells and adjust the volume of the cell suspension to fit the experiment (typically 300-500 uL). Transfer the cells to a 15 ml tube. Add 10 – 17 uL of 30X FLICA. Incubate at 37 degrees C, 30-60 minutes, mixing gently every 10 minutes. Wash by adding ~10 mL of medium and incubate at 37 °C for 60 min to allow unbound FLICA to diffuse out of cells. Spin at 220 x g for 5 min; aspirate supernatant. Add ~300 uL of 1X apoptosis wash buffer.
Adherent cells: FLICA label before trypsinization and FACS analysis
Seed 5-8 x 104 cells in a 24-well plate in a final volume of 600 uL and allow to adhere for 24 hours. Expose cells to experimental conditions. Add 1-4 uL of FLICA 150X stock concentrate and incubate for 1-3 hours at 37°C. Remove the supernatant containing round cells and set it aside in a labelled tube. Wash the adherent cell monolayer by gently adding PBS to cover the adherent cell monolayer. Remove the PBS and combine it with the cells previously reserved in step 4. Add trypsin-versene to just cover the attached cell monolayer. Allow the cells to detach and remove detached cells by adding 1 mL of cell culture medium + 20% FBS to the trypsinized cells in the wells.
Add the separated cells from the trypsinization step to the supernatant from step 4. Add 2 mL of cell culture media + 20% FBS to each tube containing trypsinized cells. Spin cells at 220 x g for 5 min. Remove the supernatant and discard it. Add 1 mL of 1x apoptosis wash buffer. Spin cells at 220 x g for 5 min. Remove the supernatant. Add 1 mL of 1x apoptosis wash buffer. Spin cells at 220 x g for 5 min. Remove the supernatant and resuspend in 300 uL of 1X apoptosis wash buffer. If desired, add 30uL of fixative. Analyze FACS immediately.
Purpose: Caspase 3, Caspase 7
Excitation / Emission: 488nm / 530nm
Flow cytometer, fluorescence microscope, fluorescence plate reader
Sample types: Cell Culture, Tissue
Preparation and Storage: Store at 2-8 degrees C
Selected information in the online data sheet is drawn from bioinformatics databases, sometimes resulting in the ambiguous or irrelevant product information. It is the customer’s responsibility to review, verify and evaluate the information to ensure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and can be updated and improved from time to time.
If the format of this essay is important to you, please request the current manual or contact our technical support team with a presales inquiry before placing an order. We will confirm the current trial details. We cannot guarantee that the sample manual posted online is the most up-to-date manual, it is intended to serve as an example only. Please refer to the instructions for use provided with the assay kit for precise details.
Small volumes of FAM-FLICA Assay Kit vials may occasionally become caught in the product vial seal during shipping and storage. If necessary, briefly centrifuge the vial in a benchtop centrifuge to dislodge any liquid on the vial cap. Certain products may require shipping with dry ice and an additional fee for dry ice may apply.
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