{"id":44552,"date":"2023-07-28T16:14:15","date_gmt":"2023-07-28T08:14:15","guid":{"rendered":"https:\/\/biopioneer.com.tw\/?p=44552"},"modified":"2024-02-19T18:04:16","modified_gmt":"2024-02-19T10:04:16","slug":"%e7%b4%b0%e8%83%9e%e4%bb%8b%e5%b0%8e%e7%9a%84%e7%b4%b0%e8%83%9e%e6%af%92%e6%80%a7%e8%9e%a2%e5%85%89%e6%aa%a2%e6%b8%ac%ef%bc%887-aad-cfse%ef%bc%89-cell-mediated-cytotoxicity-fluorometric-assay-kit","status":"publish","type":"post","link":"https:\/\/biopioneer.com.tw\/?p=44552","title":{"rendered":"\u7d30\u80de\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027\u87a2\u5149\u6aa2\u6e2c\uff087-AAD \/ CFSE\uff09| Cell-Mediated Cytotoxicity Fluorometric Assay Kit\u8ca8\u865fKA1293"},"content":{"rendered":"

\u7d30\u80de\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027\u87a2\u5149\u6aa2\u6e2c\uff087-AAD \/ CFSE\uff09|\u8ca8\u865f\u00a0KA1293<\/span><\/a><\/span><\/p>\n

Cell-Mediated Cytotoxicity Fluorometric Assay Kit<\/p>\n

<\/p>\n

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.<\/p>\n

\u7522\u54c1\u63cf\u8ff0\uff1a<\/strong>\u7d30\u80de\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027\u662f\u4e00\u7a2e\u91cd\u8981\u73fe\u8c61\uff0c\u5176\u7279\u5fb5\u5728\u65bc\u514d\u75ab\u7cfb\u7d71\u5c0d\u9ad4\u5167\u53d7\u640d\u7d30\u80de\u7684\u7d30\u80de\u6eb6\u89e3(cytolysis)\u3002 \u514d\u75ab\u7cfb\u7d71\u6fc0\u6d3b\uff0c\u53bb\u9664\u88ab\u75c5\u539f\u9ad4\u6216\u8f49\u5316\u7d30\u80de\/\u764c\u7d30\u80de\u611f\u67d3\u7684\u9776\u7d30\u80de\u3002 \u8a72\u904e\u7a0b\u7531\u6297\u9ad4\u4f9d\u8cf4\u6027\u7d30\u80de\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027\uff08antibody-dependent cell-mediated cytotoxicity , ADCC\uff09\uff0c\u88dc\u9ad4\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027(complement-mediated cytotoxicity)\u6216\u6dcb\u5df4\u7d30\u80de\u4ecb\u5c0e(lymphocyte-mediated cytotoxicity)\u7684\u7d30\u80de\u6bd2\u6027\u4ecb\u5c0e\u3002 Abnova\u7684Cell-Mediated Cytotoxicity Assay Kit\u7d30\u80de\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027\u6aa2\u6e2c\u8a66\u5291\u76d2\uff0c\u63a1\u7528 CFSE \u6a19\u8a18\u6df7\u5408\u7d30\u80de\u5167\u7684\u9776\u7d30\u80de \u7fa4\u9ad4\u548c 7-AAD \u4f86\u6a19\u8a18\u6b7b\u7d30\u80de\u3002<\/span>\u5305\u542b\u7fa7\u57fa\u6c1f\u7425\u73c0\u9170\u4e9e\u80fa\u916f\uff08carboxyfluorosuccinimide ester , CFSE\uff09\uff0c\u4e00\u7a2e\u6a19\u8a18\u6d3b\u9776\u7d30\u80de\u7684\u7da0\u8272\u87a2\u5149\u63a2\u91dd\u548c7-\u6c28\u57fa\u653e\u7dda\u83cc\u7d20D\uff087-aminoactinomycin D , 7-AAD\uff09\uff0c\u4e00\u7a2e\u7ea2\u8272\u87a2\u5149\u63a2\u91dd\uff0c\u53ef\u6a19\u8a18\u7d30\u80de\u6bd2\u6027\u4e2d\u6b7b\u4ea1\u7684\u665a\u671f\u51cb\u4ea1\u548c\u58de\u6b7b\u9776\u7d30\u80de\u6aa2\u6e2c\u30027-AAD\/CFSE \u7d30\u80de\u4ecb\u5c0e\u7684\u7d30\u80de\u6bd2\u6027\u6aa2\u6e2c\u8a66\u5291\u76d2\u662f\u4e00\u7a2e\u7192\u5149\u6a19\u8a18\u65b9\u6cd5\uff0c\u7528\u65bc\u5728\u55ae\u7d30\u80de\u6c34\u5e73\u6e2c\u5b9a\u514d\u75ab\u6548\u61c9\u7d30\u80de\u7684\u7d30\u80de\u6bd2\u6027\u4f5c\u7528\u3002\u8a72\u8a66\u5291\u76d2\u63d0\u4f9b\u8db3\u5920\u7684\u8a66\u5291\u4f86\u6709\u6548\u67d3\u8272\u7d04 109<\/sup> \u500b\u7d30\u80de\u3002<\/span><\/p>\n

<\/span><\/p>\n

<\/p>\n

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\u00b0C. 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.<\/p>\n

\"\u7db2\u7ad9\u898f\u683c\u5c0f\u5716_\u5de5\u4f5c\u5340\u57df<\/a><\/p>\n

<\/p>\n

Assay Procedure<\/span><\/strong><\/span>
\n1. Collect effector cells into tubes. Centrifuge the cells at 400 x g for five minutes to pellet.
\n2. Resuspend the cells in culture medium at a concentration of 5 x 106 cells\/mL.
\n3. Add effector cells to the CFSE-labeled target cell suspension at a predetermined effector\/target cell ratio.
\nSome examples are shown in the table below:<\/p>\n

<\/p>\n

4. Incubate the cell mixture for four hours or for a period of time according to your optimal protocol, allowing
\nenough time for cytolytic activity to progress.
\n5. To stain in a 96 well v-bottom plate as described here, transfer cells into the plate and centrifuge at 400 x
\ng for five minutes. (For staining in tubes, scale volumes up 5-fold).
\n6. Aspirate the supernatant. Note: If additional surface markers are to be assayed, staining can be inserted
\nat this point in the protocol.
\n7. Resuspend the cells in 100 \u03bcL of 7-AAD Viability Dye (1,000X) (Prepare as described on page 4). The
\ncontrol target cells (target cells without CFSE or 7-AAD viability dye or target cells with CFSE staining
\nonly) should be resuspended in 100 \u03bcL of Assay Buffer.
\n8. Incubate the cells for 15 minutes in the dark at 4\u00b0C.
\n9. Centrifuge at 400 x g for five minutes and aspirate the supernatant.
\n10. Resuspend the cells in 200 \u2013 500 \u03bcL of Assay Buffer.
\n11. The cells are now ready for analysis with a flow cytometer and should be analyzed immediately. Gate on
\nCFSE+ target cells (ex\/em 488\/525), and then visualize the live\/dead cell percentages by 7-ADD exclusion (ex\/em 488\/647).<\/p>\n

 <\/p>\n","protected":false},"excerpt":{"rendered":"

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