細胞介導的細胞毒性螢光檢測（7-AAD / CFSE）|貨號 KA1293

Cell-Mediated Cytotoxicity Fluorometric Assay Kit

7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit is a fluorescence-labeled method for determining the cytotoxic effect of immune effector cells at a single cell level.

產品描述：細胞介導的細胞毒性是一種重要現象，其特徵在於免疫系統對體內受損細胞的細胞溶解(cytolysis)。 免疫系統激活，去除被病原體或轉化細胞/癌細胞感染的靶細胞。 該過程由抗體依賴性細胞介導的細胞毒性（antibody-dependent cell-mediated cytotoxicity , ADCC），補體介導的細胞毒性(complement-mediated cytotoxicity)或淋巴細胞介導(lymphocyte-mediated cytotoxicity)的細胞毒性介導。 Abnova的Cell-Mediated Cytotoxicity Assay Kit細胞介導的細胞毒性檢測試劑盒，採用 CFSE 標記混合細胞內的靶細胞 群體和 7-AAD 來標記死細胞。包含羧基氟琥珀酰亞胺酯（carboxyfluorosuccinimide ester , CFSE），一種標記活靶細胞的綠色螢光探針和7-氨基放線菌素D（7-aminoactinomycin D , 7-AAD），一種红色螢光探針，可標記細胞毒性中死亡的晚期凋亡和壞死靶細胞檢測。7-AAD/CFSE 細胞介導的細胞毒性檢測試劑盒是一種熒光標記方法，用於在單細胞水平測定免疫效應細胞的細胞毒性作用。該試劑盒提供足夠的試劑來有效染色約 10⁹ 個細胞。

The degree of cytotoxicity of natural killer cells (effector cells; NK cells) on K-562 cells (target cells) depends on the ratio of NK cells to K-562 cells. K-562 cells were labeled with CFSE and co-cultured with NK cells at the ratios indicated above. Cells were co-cultured for four hours at 37°C. At the end of the experiment, dead cells were labeled with 7-AAD. The percentage of K-562 cell death was analyzed by a flow cytometer. When K-562 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 K-562 cell death (8.2%). At a ratio of NK cells to target cells of 25 : 1, there is 19.7% death of K-562 cells.

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 106 cells/mL.
3. Add effector cells to the CFSE-labeled target cell suspension at a predetermined effector/target cell ratio.
Some examples are shown in the table below:

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.
5. To stain in a 96 well v-bottom plate as described here, transfer cells into the plate and centrifuge at 400 x
g for five minutes. (For staining in tubes, scale volumes up 5-fold).
6. Aspirate the supernatant. Note: If additional surface markers are to be assayed, staining can be inserted
at this point in the protocol.
7. Resuspend the cells in 100 μL of 7-AAD Viability Dye (1,000X) (Prepare as described on page 4). The
control target cells (target cells without CFSE or 7-AAD viability dye or target cells with CFSE staining
only) should be resuspended in 100 μL of Assay Buffer.
8. Incubate the cells for 15 minutes in the dark at 4°C.
9. Centrifuge at 400 x g for five minutes and aspirate the supernatant.
10. Resuspend the cells in 200 – 500 μL of Assay Buffer.
11. The cells are now ready for analysis with a flow cytometer and should be analyzed immediately. Gate on
CFSE+ target cells (ex/em 488/525), and then visualize the live/dead cell percentages by 7-ADD exclusion (ex/em 488/647).