ab AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit

Transcription

1 ab AAD/CFSE Cell- Mediated Cytotoxicity Assay Kit Instructions for Use To quantify the cytotoxic effect of immune effector cells at a single cell level. This product is for research use only and is not intended for diagnostic use.

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3 Table of Contents 1. Overview 3 2. Background 3 3. Components and Storage 5 4. Assay Protocol 6 5. Data Analysis Troubleshooting 13 2

4 1. Overview ab employs CFSE to label target cells and 7-AAD to label dead cells. This kit provides a convenient tool to quantify the cytotoxic effect of immune effector cells at a single cell level. The kit provides sufficient reagents to effectively treat/stain 10 plates worth of cells irrespective of the number of wells per plate. 2. Background Cell-mediated cytotoxicity is characterized by cytolysis of a target cell by effector cells such as cytotoxic T lymphocytes or natural killer cells. It is a major part of the immune system in which the body protects itself from attack by invading pathogens and is fundamental in maintaining homeostasis of the immune system. Dysfunction of cytotoxic T lymphocytes or natural killer cells results in pathological conditions such as multiple sclerosis and cancer. Cell-mediated cytotoxicity is a complex, multistep process which is mediated by several different pathways, including the granule exocytosis pathway and the Fas pathway. The interplay between these pathways provides opportunities for therapeutic interventions to control autoimmune diseases and graft versus host disease. Cell-mediated cytotoxicity has typically been assessed using the 51 chromium ( 51 Cr) release assay, in which the target cells are labeled with 51 Cr. This method has disadvantages including the use of radioisotopes and low sensitivity. 3

5 More sensitive and convenient methods have been developed using green fluorescent probes such as 5-(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to label target cells, in combination with red fluorescent probes such as 7-AAD or propidium iodide to label dead effector and dead target cells. CFSE passively diffuses into cells and its acetate groups are cleaved by intracellular esterases to yield highly fluorescent carboxyfluorescein succinimidyl ester. 7-AAD and propidium iodide are fluorescent dyes which are excluded from live cells but penetrate dead or damaged cells to label DNA. The labeling distinguishes four populations of cells: 1) living target cells in green, 2) dead target cells in green and red, 3) dead effector cells in red, and 4) live effector cells, which remain unstained. Thus, the cytotoxicity of the effector cells can be evaluated quantitatively. 4

6 3. Components and Storage For best results, remove components and store as stated in table. Item Quantity Storage Cell-Based Assay 7-AAD Staining Stock Solution (1,000X) 2 vials/50 μl 4 C CFSE Stock Solution 1 vial/100 μl -20 C Cell-Based Assay Buffer Tablet 4 Tablets Room Temperature Materials Needed But Not Supplied A 6-, 12-, or 24-well plate for culturing cells 2. A flow cytometer equipped with a 488 nm excitation laser 3. Bovine serum albumin (BSA) 5

7 4. Assay Protocol A. Preparation of Assay-Specific Reagents Cell-Based Assay Buffer Preparation Dissolve each Cell-Based Assay Buffer Tablet with 100 ml of distilled water. Mix well to ensure that the tablet dissolves completely. The diluted buffer is stable at room temperature for one year. 7-AAD Staining Solution Preparation Add 10 μl of Cell-Based Assay 7-AAD Staining Stock Solution (1,000X) to 10 ml of Assay Buffer and mix well. CFSE Staining Solution Preparation First, prepare a 0.1% BSA/Assay Buffer by adding 10 mg BSA to 10 ml of Assay Buffer. Then add 10 μl of CFSE Stock Solution to 10 ml of 0.1% BSA/Assay Buffer and mix well. 6

8 B. Performing the assay To properly analyze the data, the following control target cell groups are needed to set up the flow cytometer and compensation: Target cells without CFSE or 7-AAD staining Target cells with CFSE staining only Target cells with 7-AAD staining only Target cells with both CFSE and 7-AAD staining Labeling of target cells 1. Seed the target cells in a 6-, 12-, or 24-well plate at a density of cells/well in 2, 1, or 0.5 ml of culture medium, respectively. Culture the cells in a CO 2 incubator at 37 C for at least 24 hours before treatment. 2. Collect the cells into tubes. Centrifuge the cells at 400 x g for five minutes to pellet the cells and wash once with 1 ml of Assay Buffer. 3. Resuspend cell pellet in CFSE Staining Solution at a concentration of 10 6 cells/ml. Control target cells (target cells without CFSE or 7-AAD staining or target cells with 7-AAD staining only) should be resuspended in 0.1% BSA/Assay Buffer. 7

9 4. Vortex target cells gently. Incubate the cells in the CFSE Staining Solution for 15 minutes at room temperature. 5. Centrifuge the target cells at 400 x g for five minutes. 6. Aspirate the supernatant. 7. Resuspend the target cells in the culture medium at a concentration of 10 6 cells/ml. 8. Centrifuge the target cells at 400 x g for five minutes. 9. Aspirate the supernatant. 10. Resuspend the target cells in culture medium at a concentration of 10 5 cells/ml. 11. Incubate the cells at 37 C for 30 minutes or longer (but not long enough for the cells to proliferate) in a CO 2 incubator. 8

10 C. Assay procedure 1. Collect effector cells into tubes. Centrifuge the cells at 400 x g for five minutes to pellet. 2. Resuspend the cells in culture medium at a concentration of 5 x 10 6 cells/ml. 3. Add effector cells to the CFSE-labeled target cell suspension at a predetermined effector/target cell ratio. The four control groups of target cells received no effector cells. Some examples are shown in Table 1. Effector/ Effector Cell Target Cell Target Cell Final Target Ratio Suspension Suspension Medium volume 0 0 ml 1.5 ml 0 ml 1.5 ml 6.25: ml 1 ml ml 1.5 ml 12.5: ml 1 ml 0.25 ml 1.5 ml 25:1 0.5 ml 1 ml 0 ml 1.5 ml Table 1. Addition of effector cells to Target cell suspension 4. Incubate the cell mixture for four hours or for a period of time according to your optimal protocol, allowing enough time for cytolytic activity to progress. 9

11 5. Centrifuge the cell mixture to pellet the cells. 6. Aspirate the supernatant. 7. Resuspend the cells in 0.5 ml-1 ml of 7-AAD Staining Solution and mix well. The control target cells (target cells without CFSE or 7-AAD staining or target cells with CFSE staining only) should be resuspended in ml of Assay Buffer. 8. Incubate the cells for 15 minutes in the dark at 4 C. 9. Centrifuge the cell mixture to pellet the cells. 10. Resuspend the cells in ml of Assay Buffer. 11. The cells are now ready for analysis with a flow cytometer and should be analyzed immediately. CFSE cell staining is measured in the FL1 channel and 7-AAD cell staining is measured in the FL3 channel. To ensure that only proper target cells are gated, use a side scatter versus FL-1 plot. 10

12 5. Data Analysis Performance Characteristics An example of typical data obtained using flow cytometry is shown in the figure 1. Your results may vary based on the cell type and experimental protocol used. 11

13 Figure 1. The degree of cytotoxicity of natural killer cells (effector cells; NK cells) on K562 cells (target cells) depends on the ratio of NK cells to K562 cells. K562cells were labeled with CFSE and cocultured with NK cells at the ratios indicated above. Cells were cocultured for four hours at 37 C. At the end of the experiment, dead cells were labeled with 7-AAD. The percentage of K562 cell death was analyzed by a flow cytometer. When K562 cells were cultured alone, there is little cell death (2%). Addition of NK cells at a ratio as low as 6.25:1 caused a significant increase in K562 cell death (8.2%). At a ratio of NK cells to target cells of 25:1, there is 19.7% death of K562 cells. 12

14 6. Troubleshooting Problem Possible Causes Recommended Solutions Low signal of CFSE No difference in cytotoxicity among different effector cell to target cell ratios A. Cells are not healthy B. Cells were not well labeled by CFSE. A. Target cells are not healthy B. Effector cells do not cause cytotoxicity A. Use only healthy cells B. Perform a titration of CFSE to get an optimal concentration of CFSE staining. A. Use only healthy target cells B. Use effector cells that cause cytotoxicity 13

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