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\u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd \u0b85\u0bb2\u0bcd\u0bb2\u0ba4\u0bc1 \u0b89\u0b9f\u0ba9\u0b9f\u0bbf\u0baf\u0bbe\u0b95 \u0b95\u0bb2\u0bbe\u0b9a\u0bcd\u0b9a\u0bbe\u0bb0\u0b99\u0bcd\u0b95\u0bb3\u0bc8\u0ba4\u0bcd \u0ba4\u0bca\u0b9f\u0b99\u0bcd\u0b95\u0bb5\u0bbf\u0bb2\u0bcd\u0bb2\u0bc8 \u0b8e\u0ba9\u0bcd\u0bb1\u0bbe\u0bb2\u0bcd \u0ba4\u0bbf\u0bb0\u0bb5 \u0ba8\u0bc8\u0b9f\u0bcd\u0bb0\u0b9c\u0ba9\u0bcd \u0b9a\u0bc7\u0bae\u0bbf\u0baa\u0bcd\u0baa\u0b95\u0ba4\u0bcd\u0ba4\u0bbf\u0bb2\u0bcd \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd. \u0b87\u0bb0\u0bc1\u0baa\u0bcd\u0baa\u0bbf\u0ba9\u0bc1\u0bae\u0bcd, \u0ba8\u0bc0\u0b99\u0bcd\u0b95\u0bb3\u0bcd \u0baa\u0bbf\u0ba9\u0bcd\u0ba9\u0bb0\u0bcd \u0baa\u0baf\u0ba9\u0bcd\u0baa\u0b9f\u0bc1\u0ba4\u0bcd\u0ba4 \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8\u0b9a\u0bcd \u0b9a\u0bc7\u0bae\u0bbf\u0b95\u0bcd\u0b95\u0baa\u0bcd \u0baa\u0bcb\u0b95\u0bbf\u0bb1\u0bc0\u0bb0\u0bcd\u0b95\u0bb3\u0bcd \u0b8e\u0ba9\u0bcd\u0bb1\u0bbe\u0bb2\u0bcd, \u0baa\u0bc1\u0ba4\u0bbf\u0baf \u0baa\u0b99\u0bcd\u0b95\u0bc1\u0b95\u0bb3\u0bc8 \u0bb5\u0bb3\u0bb0\u0bcd\u0ba4\u0bcd\u0ba4\u0bc1, \u0baa\u0bb2 \u0b86\u0bae\u0bcd\u0baa\u0bc2\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0b89\u0bb1\u0bc8\u0baf \u0bb5\u0bc8\u0b95\u0bcd\u0b95 \u0baa\u0bb0\u0bbf\u0ba8\u0bcd\u0ba4\u0bc1\u0bb0\u0bc8\u0b95\u0bcd\u0b95\u0baa\u0bcd\u0baa\u0b9f\u0bc1\u0b95\u0bbf\u0bb1\u0ba4\u0bc1. \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd 45% RPMI-1640, 50% FBS \u0bae\u0bb1\u0bcd\u0bb1\u0bc1\u0bae\u0bcd 5% DMSO (\u0ba4\u0bbf\u0b9a\u0bc1 \u0bb5\u0bb3\u0bb0\u0bcd\u0baa\u0bcd\u0baa\u0bc1 \u0ba4\u0bb0\u0bae\u0bcd) \u0b86\u0b95\u0bbf\u0baf\u0bb5\u0bb1\u0bcd\u0bb1\u0bbf\u0bb2\u0bcd \u0b95\u0bbf\u0bb0\u0bc8\u0baf\u0bcb\u0baa\u0bbf\u0bb0\u0bc7\u0b9a\u0bb0\u0bcd\u0bb5\u0bcd \u0b9a\u0bc6\u0baf\u0bcd\u0baf\u0baa\u0bcd\u0baa\u0b9f\u0bc1\u0b95\u0bbf\u0ba9\u0bcd\u0bb1\u0ba9.<\/p>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>\u0b95\u0bbe\u0bb1\u0bcd\u0bb1\u0bcb\u0b9f\u0bcd\u0b9f\u0bae\u0bbe\u0ba9 \u0bae\u0bc2\u0b9f\u0bbf\u0baf\u0bc1\u0b9f\u0ba9\u0bcd \u0b95\u0bc2\u0b9f\u0bbf\u0baf T-25 \u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b95\u0bbf\u0bb2\u0bcd 10 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf RPMI-1640, 15% \u0b95\u0bb0\u0bc1 \u0baa\u0bcb\u0bb5\u0bbf\u0ba9\u0bcd \u0b9a\u0bc0\u0bb0\u0bae\u0bcd (FBS), \u00b1 \u0ba8\u0bc1\u0ba3\u0bcd\u0ba3\u0bc1\u0baf\u0bbf\u0bb0\u0bcd \u0b8e\u0ba4\u0bbf\u0bb0\u0bcd\u0baa\u0bcd\u0baa\u0bbf\u0b95\u0bb3\u0bc8 \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>5% CO \u0b89\u0b9f\u0ba9\u0bcd 37\u00b0C \u0b88\u0bb0\u0baa\u0bcd\u0baa\u0ba4\u0bae\u0bbe\u0ba9 \u0b87\u0ba9\u0bcd\u0b95\u0bc1\u0baa\u0bc7\u0b9f\u0bcd\u0b9f\u0bb0\u0bbf\u0bb2\u0bcd \u0b8a\u0b9f\u0b95\u0ba4\u0bcd\u0ba4\u0bc8 \u0b9a\u0bae\u0ba8\u0bbf\u0bb2\u0bc8\u0baa\u0bcd\u0baa\u0b9f\u0bc1\u0ba4\u0bcd\u0ba4 \u0b85\u0ba9\u0bc1\u0bae\u0ba4\u0bbf\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<sub>2<\/sub> 30 \u0ba8\u0bbf\u0bae\u0bbf\u0b9f\u0b99\u0bcd\u0b95\u0bb3\u0bcd \u0b95\u0bbe\u0bb1\u0bcd\u0bb1\u0bbf\u0bb2\u0bcd \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd. \u0b95\u0bc1\u0b9f\u0bc1\u0bb5\u0bc8\u0b95\u0bb3\u0bc8 \u0ba8\u0bbf\u0bae\u0bbf\u0bb0\u0bcd\u0ba8\u0bcd\u0ba4 \u0ba8\u0bbf\u0bb2\u0bc8\u0baf\u0bbf\u0bb2\u0bcd \u0b85\u0b9f\u0bc8\u0b95\u0bbe\u0b95\u0bcd\u0b95 \u0bb5\u0bc7\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0b89\u0bb1\u0bc8\u0ba8\u0bcd\u0ba4 \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bbf\u0ba9\u0bcd \u0b92\u0bb5\u0bcd\u0bb5\u0bca\u0bb0\u0bc1 \u0b86\u0bae\u0bcd\u0baa\u0bc2\u0bb2\u0bc8\u0baf\u0bcb \u0b85\u0bb2\u0bcd\u0bb2\u0ba4\u0bc1 \u0b95\u0bc1\u0baa\u0bcd\u0baa\u0bbf\u0baf\u0bc8\u0baf\u0bcb 37\u00b0C \u0ba8\u0bc0\u0bb0\u0bcd \u0b95\u0bc1\u0bb3\u0bbf\u0baf\u0bb2\u0bcd \u0b85\u0bb2\u0bcd\u0bb2\u0ba4\u0bc1 \u0b92\u0bb0\u0bc1 \u0baa\u0bc0\u0b95\u0bcd\u0b95\u0bb0\u0bcd \u0bb5\u0bc6\u0ba4\u0bc1\u0bb5\u0bc6\u0ba4\u0bc1\u0baa\u0bcd\u0baa\u0bbe\u0ba9 \u0ba8\u0bc0\u0bb0\u0bbf\u0bb2\u0bcd \u0b92\u0bb5\u0bcd\u0bb5\u0bca\u0ba9\u0bcd\u0bb1\u0bbe\u0b95\u0b95\u0bcd \u0b95\u0bb0\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>70% \u0b8e\u0ba4\u0bcd\u0ba4\u0ba9\u0bbe\u0bb2\u0bcd \u0b95\u0bca\u0ba3\u0bcd\u0b9f\u0bc1 \u0b86\u0bae\u0bcd\u0baa\u0bc2\u0bb2\u0bcd \u0b85\u0bb2\u0bcd\u0bb2\u0ba4\u0bc1 \u0b95\u0bc1\u0baa\u0bcd\u0baa\u0bbf\u0baf\u0bbf\u0ba9\u0bcd \u0bb5\u0bc6\u0bb3\u0bbf\u0baa\u0bcd\u0baa\u0bc1\u0bb1\u0ba4\u0bcd\u0ba4\u0bc8 \u0bb5\u0bbf\u0bb0\u0bc8\u0bb5\u0bbe\u0b95 \u0b9a\u0bc1\u0ba4\u0bcd\u0ba4\u0bae\u0bcd \u0b9a\u0bc6\u0baf\u0bcd\u0baf\u0bb5\u0bc1\u0bae\u0bcd. \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd \u0b92\u0bb0\u0bc1 \u0b95\u0ba3\u0bcd\u0ba3\u0bbe\u0b9f\u0bbf \u0b86\u0bae\u0bcd\u0baa\u0bc2\u0bb2\u0bbf\u0bb2\u0bcd \u0b87\u0bb0\u0bc1\u0ba8\u0bcd\u0ba4\u0bbe\u0bb2\u0bcd, \u0b89\u0b9f\u0bc8\u0ba8\u0bcd\u0ba4 \u0b95\u0ba3\u0bcd\u0ba3\u0bbe\u0b9f\u0bbf\u0baf\u0bbf\u0bb2\u0bbf\u0bb0\u0bc1\u0ba8\u0bcd\u0ba4\u0bc1 \u0bb5\u0bbf\u0bb0\u0bb2\u0bcd\u0b95\u0bb3\u0bc8\u0baa\u0bcd \u0baa\u0bbe\u0ba4\u0bc1\u0b95\u0bbe\u0b95\u0bcd\u0b95 \u0b86\u0bae\u0bcd\u0baa\u0bc2\u0bb2\u0bc8 \u0b95\u0bb5\u0ba9\u0bae\u0bbe\u0b95 \u0b89\u0b9f\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>1 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0b89\u0bb1\u0bc8\u0ba8\u0bcd\u0ba4 \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0b92\u0bb0\u0bc1 \u0bae\u0bb2\u0b9f\u0bcd\u0b9f\u0bc1 \u0baa\u0bc8\u0baa\u0bcd\u0baa\u0b9f\u0bcd \u0bae\u0bc2\u0bb2\u0bae\u0bcd \u0b85\u0b95\u0bb1\u0bcd\u0bb1\u0bbf, 10 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0bae\u0bc0\u0b9f\u0bbf\u0baf\u0bae\u0bcd \u0b95\u0bca\u0ba3\u0bcd\u0b9f T-25 \u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b95\u0bbf\u0bb2\u0bcd \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd. \u0b92\u0bb5\u0bcd\u0bb5\u0bca\u0bb0\u0bc1 \u0b9a\u0bc6\u0bb2\u0bcd \u0b95\u0bcb\u0b9f\u0bcd\u0b9f\u0bc8\u0baf\u0bc1\u0bae\u0bcd \u0b85\u0b9f\u0bc8\u0baf\u0bbe\u0bb3\u0bae\u0bcd \u0b95\u0bbe\u0ba3 \u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b95\u0bbf\u0bb2\u0bcd \u0ba4\u0bc6\u0bb3\u0bbf\u0bb5\u0bbe\u0b95 \u0bb2\u0bc7\u0baa\u0bbf\u0bb3\u0bbf\u0b9f\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>5% CO2 \u0b89\u0b9f\u0ba9\u0bcd 37\u00b0C \u0b88\u0bb0\u0baa\u0bcd\u0baa\u0ba4\u0bae\u0bbe\u0ba9 \u0b87\u0ba9\u0bcd\u0b95\u0bc1\u0baa\u0bc7\u0b9f\u0bcd\u0b9f\u0bb0\u0bbf\u0bb2\u0bcd \u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b95\u0bc8 \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<sub>2<\/sub> \u0b95\u0bbe\u0bb1\u0bcd\u0bb1\u0bbf\u0bb2\u0bcd. \u0b95\u0bc1\u0b9f\u0bc1\u0bb5\u0bc8\u0b95\u0bb3\u0bc8 \u0ba8\u0bbf\u0bae\u0bbf\u0bb0\u0bcd\u0ba8\u0bcd\u0ba4 \u0ba8\u0bbf\u0bb2\u0bc8\u0baf\u0bbf\u0bb2\u0bcd \u0b85\u0b9f\u0bc8\u0b95\u0bbe\u0b95\u0bcd\u0b95 \u0bb5\u0bc7\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd.<\/li>\n<li>24 \u0bae\u0ba3\u0bbf \u0ba8\u0bc7\u0bb0\u0ba4\u0bcd\u0ba4\u0bbf\u0bb1\u0bcd\u0b95\u0bc1\u0baa\u0bcd \u0baa\u0bbf\u0bb1\u0b95\u0bc1, \u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b95\u0bbf\u0ba9\u0bcd \u0b85\u0b9f\u0bbf\u0baa\u0bcd\u0baa\u0b95\u0bc1\u0ba4\u0bbf\u0baf\u0bbf\u0bb2\u0bcd \u0b95\u0bc1\u0b9f\u0bbf\u0baf\u0bc7\u0bb1\u0bbf\u0baf \u0bb2\u0bbf\u0bae\u0bcd\u0baa\u0bcb\u0b9a\u0bc8\u0b9f\u0bcd\u0b9f\u0bc1\u0b95\u0bb3\u0bc8\u0ba4\u0bcd \u0ba4\u0bca\u0ba8\u0bcd\u0ba4\u0bb0\u0bb5\u0bc1 \u0b9a\u0bc6\u0baf\u0bcd\u0baf\u0bbe\u0ba4 \u0bb5\u0b95\u0bc8\u0baf\u0bbf\u0bb2\u0bcd \u0bae\u0bc7\u0bb2\u0bbf\u0bb0\u0bc1\u0ba8\u0bcd\u0ba4\u0bc1 5 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0bae\u0bc0\u0b9f\u0bbf\u0baf\u0ba4\u0bcd\u0ba4\u0bc8 \u0b85\u0b95\u0bb1\u0bcd\u0bb1\u0bbf, \u0bae\u0bc1\u0ba9\u0bcd \u0b9a\u0bc2\u0b9f\u0bbe\u0b95\u0bcd\u0b95\u0baa\u0bcd\u0baa\u0b9f\u0bcd\u0b9f 5 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0bae\u0bc0\u0b9f\u0bbf\u0baf\u0ba4\u0bcd\u0ba4\u0bc1\u0b9f\u0ba9\u0bcd \u0bae\u0bbe\u0bb1\u0bcd\u0bb1\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0bb2\u0bbf\u0bae\u0bcd\u0baa\u0bcb\u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b9f\u0bcd \u0b9a\u0bc6\u0bb2\u0bcd \u0bb5\u0bb3\u0bb0\u0bcd\u0baa\u0bcd\u0baa\u0bc1 \u0ba8\u0bc6\u0bb1\u0bbf\u0bae\u0bc1\u0bb1\u0bc8\u0baf\u0bc1\u0b9f\u0ba9\u0bcd \u0ba4\u0bca\u0b9f\u0bb0\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<h4><strong><\/strong><\/h4>\n<h4><strong>\u0bb2\u0bbf\u0bae\u0bcd\u0baa\u0bcb\u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b9f\u0bcd \u0b95\u0bb2\u0bbe\u0b9a\u0bcd\u0b9a\u0bbe\u0bb0\u0bae\u0bcd<\/strong><\/h4>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>\u0b89\u0bb0\u0bc1\u0b95\u0bbf\u0baf \u0bae\u0bc2\u0ba9\u0bcd\u0bb1\u0bc1 \u0bae\u0bc1\u0ba4\u0bb2\u0bcd \u0ba8\u0bbe\u0ba9\u0bcd\u0b95\u0bc1 \u0ba8\u0bbe\u0b9f\u0bcd\u0b95\u0bb3\u0bc1\u0b95\u0bcd\u0b95\u0bc1\u0baa\u0bcd \u0baa\u0bbf\u0bb1\u0b95\u0bc1, \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0b8e\u0ba3\u0bcd\u0ba3\u0bc1\u0b99\u0bcd\u0b95\u0bb3\u0bcd. \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd \u0b92\u0b9f\u0bcd\u0b9f\u0bbe\u0bae\u0bb2\u0bcd \u0b87\u0bb0\u0bc1\u0b95\u0bcd\u0b95\u0bc1\u0bae\u0bcd, \u0b95\u0bca\u0ba4\u0bcd\u0ba4\u0bbe\u0b95 \u0bb5\u0bb3\u0bb0\u0bc1\u0bae\u0bcd. \u0bae\u0bc6\u0ba9\u0bcd\u0bae\u0bc8\u0baf\u0bbe\u0ba9 \u0b95\u0bc1\u0bb4\u0bbe\u0baf\u0bcd \u0baa\u0ba4\u0bbf\u0ba4\u0bcd\u0ba4\u0bb2\u0bcd \u0bae\u0bc2\u0bb2\u0bae\u0bcd \u0b95\u0bca\u0ba4\u0bcd\u0ba4\u0bbe\u0b95 \u0b89\u0b9f\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0b8e\u0ba3\u0bcd\u0ba3\u0bc1\u0b99\u0bcd\u0b95\u0bb3\u0bcd.<\/li>\n<li>\u0bb5\u0bc6\u0bb1\u0bc1\u0bae\u0ba9\u0bc7, \u0b9a\u0bc6\u0bb2\u0bcd \u0b9a\u0bc6\u0bb1\u0bbf\u0bb5\u0bc1 2 x 10 \u0b90 \u0bb5\u0bbf\u0b9f \u0b85\u0ba4\u0bbf\u0b95\u0bae\u0bbe\u0b95 \u0b87\u0bb0\u0bc1\u0b95\u0bcd\u0b95\u0b95\u0bcd\u0b95\u0bc2\u0b9f\u0bbe\u0ba4\u0bc1.<sup>6<\/sup> \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd\/\u0bae\u0bbf.\u0bb2\u0bbf.<\/li>\n<li>\u0b9a\u0bc6\u0bb2\u0bcd \u0b9a\u0bc6\u0bb1\u0bbf\u0bb5\u0bc8\u0baa\u0bcd \u0baa\u0bca\u0bb1\u0bc1\u0ba4\u0bcd\u0ba4\u0bc1 \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0bae\u0bc0\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd \u0b8a\u0b9f\u0bcd\u0b9f\u0bb5\u0bc1\u0bae\u0bcd, \u0baa\u0bbf\u0bb0\u0bbf\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd \u0b85\u0bb2\u0bcd\u0bb2\u0ba4\u0bc1 \u0b89\u0bb1\u0bc8\u0baf \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>5-6 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0b9a\u0bae\u0baa\u0bcd\u0baa\u0b9f\u0bc1\u0ba4\u0bcd\u0ba4\u0baa\u0bcd\u0baa\u0b9f\u0bcd\u0b9f \u0bb5\u0bb3\u0bb0\u0bcd\u0b9a\u0bcd\u0b9a\u0bbf \u0b8a\u0b9f\u0b95\u0ba4\u0bcd\u0ba4\u0bc8\u0b9a\u0bcd \u0b9a\u0bc7\u0bb0\u0bcd\u0ba4\u0bcd\u0ba4\u0bc1 \u0bae\u0bc0\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd \u0b89\u0ba3\u0bb5\u0bb3\u0bbf\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>2 x 10 \u0b95\u0bcd\u0b95\u0bc1\u0bae\u0bcd \u0b95\u0bc1\u0bb1\u0bc8\u0baf\u0bbe\u0bae\u0bb2\u0bcd \u0baa\u0bc1\u0ba4\u0bbf\u0baf \u0baa\u0baf\u0bbf\u0bb0\u0bcd\u0b95\u0bb3\u0bc8 \u0bb5\u0bbf\u0ba4\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<sup>5<\/sup> \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd\/\u0bae\u0bbf.\u0bb2\u0bbf.<\/li>\n<li>\u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd \u0ba4\u0bcb\u0bb0\u0bbe\u0baf\u0bae\u0bbe\u0b95 5 x 10 \u0b87\u0bb2\u0bcd \u0b89\u0bb1\u0bc8\u0baf \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0baa\u0bcd\u0baa\u0b9f \u0bb5\u0bc7\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd.<sup>6<\/sup> \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd\/\u0bae\u0bbf\u0bb2\u0bbf. \u0b9a\u0bc6\u0bb2\u0bcd \u0b89\u0bb1\u0bc8\u0ba4\u0bb2\u0bcd \u0ba8\u0bc6\u0bb1\u0bbf\u0bae\u0bc1\u0bb1\u0bc8\u0baf\u0bc8\u0baa\u0bcd \u0baa\u0bbf\u0ba9\u0bcd\u0baa\u0bb1\u0bcd\u0bb1\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<h4><strong><\/strong><\/h4>\n<h4><strong>\u0b89\u0bb1\u0bc8\u0baa\u0ba9\u0bbf \u0bb2\u0bbf\u0bae\u0bcd\u0baa\u0bcb\u0baa\u0bbf\u0bb3\u0bbe\u0bb8\u0bcd\u0b9f\u0bcd\u0b95\u0bb3\u0bcd<\/strong><\/h4>\n<ol>\n<li style=\"list-style-type: none;\">\n<ol>\n<li>\u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd \u0ba4\u0bcb\u0bb0\u0bbe\u0baf\u0bae\u0bbe\u0b95 5 x 10 \u0b87\u0bb2\u0bcd \u0b89\u0bb1\u0bc8\u0baf \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0baa\u0bcd\u0baa\u0b9f \u0bb5\u0bc7\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd.<sup>6<\/sup> \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bcd\/\u0bae\u0bbf.\u0bb2\u0bbf.<\/li>\n<li>15 \u0b85\u0bb2\u0bcd\u0bb2\u0ba4\u0bc1 50 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0b95\u0bc2\u0bae\u0bcd\u0baa\u0bc1 \u0bb5\u0b9f\u0bbf\u0bb5\u0b95\u0bcd \u0b95\u0bc1\u0bb4\u0bbe\u0baf\u0bbf\u0bb2\u0bcd \u0baa\u0bca\u0bb0\u0bc1\u0ba4\u0bcd\u0ba4\u0bae\u0bbe\u0ba9 \u0b85\u0bb3\u0bb5\u0bbf\u0bb2\u0bbe\u0ba9 \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0bae\u0bbe\u0bb1\u0bcd\u0bb1\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>100 xg \u0b87\u0bb2\u0bcd 4\u00b0C \u0b87\u0bb2\u0bcd \u0b9a\u0bc6\u0ba9\u0bcd\u0b9f\u0bcd\u0bb0\u0bbf\u0b83\u0baa\u0bbf\u0baf\u0bc2\u0b9c\u0bc8 10 \u0ba8\u0bbf\u0bae\u0bbf\u0b9f\u0b99\u0bcd\u0b95\u0bb3\u0bc1\u0b95\u0bcd\u0b95\u0bc1 \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0bae\u0bc0\u0b9f\u0bbf\u0baf\u0ba4\u0bcd\u0ba4\u0bc8 \u0b85\u0b95\u0bb1\u0bcd\u0bb1\u0bbf, \u0baa\u0bca\u0bb0\u0bc1\u0ba4\u0bcd\u0ba4\u0bae\u0bbe\u0ba9 \u0b85\u0bb3\u0bb5\u0bc1 \u0b95\u0bc1\u0bb3\u0bbf\u0bb0\u0bcd \u0b89\u0bb1\u0bc8\u0baa\u0ba9\u0bbf \u0bae\u0bc0\u0b9f\u0bbf\u0baf\u0ba4\u0bcd\u0ba4\u0bbf\u0bb2\u0bcd \u0bae\u0bc0\u0ba3\u0bcd\u0b9f\u0bc1\u0bae\u0bcd \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0b92\u0bb5\u0bcd\u0bb5\u0bca\u0ba9\u0bcd\u0bb1\u0bc1\u0bae\u0bcd 1 \u0bae\u0bbf\u0bb2\u0bcd\u0bb2\u0bbf \u0b9a\u0bc6\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0b95\u0bbf\u0bb0\u0bc8\u0baf\u0bcb\u0bb5\u0bbf\u0baf\u0bb2\u0bc1\u0b95\u0bcd\u0b95\u0bc1 \u0bae\u0bbe\u0bb1\u0bcd\u0bb1\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0b95\u0bbf\u0bb0\u0bc8\u0baf\u0bcb\u0bb5\u0bbf\u0baf\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0b89\u0bb1\u0bc8\u0bb5\u0bbf\u0baa\u0bcd\u0baa\u0bbe\u0ba9\u0bcd \u0b85\u0bb1\u0bc8\u0baf\u0bbf\u0bb2\u0bcd \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd, \u0b87\u0bb0\u0bb5\u0bc1 \u0bae\u0bc1\u0bb4\u0bc1\u0bb5\u0ba4\u0bc1\u0bae\u0bcd -80\u00b0C \u0bb5\u0bc6\u0baa\u0bcd\u0baa\u0ba8\u0bbf\u0bb2\u0bc8\u0baf\u0bbf\u0bb2\u0bcd \u0b89\u0bb1\u0bc8\u0bb5\u0bbf\u0baa\u0bcd\u0baa\u0bbe\u0ba9\u0bcd \u0baa\u0bc6\u0b9f\u0bcd\u0b9f\u0bbf\u0baf\u0bbf\u0bb2\u0bcd \u0bb5\u0bc8\u0b95\u0bcd\u0b95\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<li>\u0bae\u0bb1\u0bc1\u0ba8\u0bbe\u0bb3\u0bcd \u0b95\u0bbf\u0bb0\u0bc8\u0baf\u0bcb\u0bb5\u0bbf\u0baf\u0bb2\u0bcd\u0b95\u0bb3\u0bc8 \u0ba4\u0bbf\u0bb0\u0bb5 \u0ba8\u0bc8\u0b9f\u0bcd\u0bb0\u0b9c\u0ba9\u0bc1\u0b95\u0bcd\u0b95\u0bc1 \u0bae\u0bbe\u0bb1\u0bcd\u0bb1\u0bb5\u0bc1\u0bae\u0bcd.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner _builder_version=&#8221;4.27.4&#8243; _module_preset=&#8221;default&#8221; hover_enabled=&#8221;0&#8243; global_colors_info=&#8221;{}&#8221; sticky_enabled=&#8221;0&#8243; custom_padding=&#8221;24px||24px|||&#8221;][et_pb_column_inner saved_specialty_column_type=&#8221;3_4&#8243; _builder_version=&#8221;4.27.4&#8243; _module_preset=&#8221;default&#8221; global_colors_info=&#8221;{}&#8221;][et_pb_button button_url=&#8221;https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2025\/02\/Lymphoblast-protocol-1-22-25.pdf&#8221; button_text=&#8221;Protocol for Culturing Lymphoblasts&#8221; admin_label=&#8221;Protocol for Culturing Lymphoblasts&#8221; _builder_version=&#8221;4.27.4&#8243; _module_preset=&#8221;default&#8221; button_text_color=&#8221;#FFFFFF&#8221; button_bg_color=&#8221;#00B2E2&#8243; button_border_color=&#8221;#00B2E2&#8243; global_colors_info=&#8221;{}&#8221;][\/et_pb_button][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=&#8221;on|phone&#8221; _builder_version=&#8221;4.16&#8243; custom_padding=&#8221;||0px|||&#8221; custom_padding_tablet=&#8221;|35px||35px||true&#8221; custom_padding_phone=&#8221;&#8221; border_width_top=&#8221;10px&#8221; border_color_top=&#8221;#8fd2ed&#8221; global_colors_info=&#8221;{}&#8221;][et_pb_column_inner saved_specialty_column_type=&#8221;3_4&#8243; _builder_version=&#8221;4.16&#8243; custom_padding=&#8221;|||&#8221; global_colors_info=&#8221;{}&#8221; custom_padding__hover=&#8221;|||&#8221;][et_pb_text _builder_version=&#8221;4.16&#8243; global_colors_info=&#8221;{}&#8221;]<\/p>\n<h4><strong>\u0bae\u0bc7\u0bb2\u0bc1\u0bae\u0bcd \u0ba4\u0b95\u0bb5\u0bb2\u0bc1\u0b95\u0bcd\u0b95\u0bc1, \u0ba4\u0bca\u0b9f\u0bb0\u0bcd\u0baa\u0bc1 \u0b95\u0bca\u0bb3\u0bcd\u0bb3\u0bb5\u0bc1\u0bae\u0bcd:<\/strong><\/h4>\n<p>[\/et_pb_text]] custom_padding=\u201d|35px|0|0px|false|false\u201d custom_padding_tablet=\u201d|35px||35px||true\u201d custom_padding_phone=\u201d\u201d animation_direction=\u201dtop\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner type=1\u2033\u2033\u201d saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|20px||\u201d custom_padding_tablet=\u201d||20px\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|desktop\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.10=9.5\u2033\u201d<\/p>\n<h6>\u0bb2\u0bc6\u0bb8\u0bcd\u0bb2\u0bbf \u0baa\u0bbf. \u0b95\u0bbe\u0bb0\u0bcd\u0b9f\u0ba9\u0bcd, MD, PhD<\/h6>\n<p>\u0baa\u0bbf\u0bb0\u0bb5\u0bc1\u0ba9\u0bcd \u0baa\u0bb2\u0bcd\u0b95\u0bb2\u0bc8\u0b95\u0bcd\u0b95\u0bb4\u0b95\u0ba4\u0bcd\u0ba4\u0bbf\u0ba9\u0bcd \u0b95\u0bc1\u0bb4\u0ba8\u0bcd\u0ba4\u0bc8 \u0bae\u0bb0\u0bc1\u0ba4\u0bcd\u0ba4\u0bc1\u0bb5 \u0b86\u0bb0\u0bbe\u0baf\u0bcd\u0b9a\u0bcd\u0b9a\u0bbf \u0baa\u0bc7\u0bb0\u0bbe\u0b9a\u0bbf\u0bb0\u0bbf\u0baf\u0bb0\u0bcd \u0bb5\u0bbe\u0bb0\u0ba9\u0bcd \u0b86\u0bb2\u0bcd\u0baa\u0bb0\u0bcd\u0b9f\u0bcd \u0bae\u0bb0\u0bc1\u0ba4\u0bcd\u0ba4\u0bc1\u0bb5\u0baa\u0bcd \u0baa\u0bb3\u0bcd\u0bb3\u0bbf \u0bae\u0bb1\u0bcd\u0bb1\u0bc1\u0bae\u0bcd \u0b95\u0bc1\u0bb4\u0ba8\u0bcd\u0ba4\u0bc8 \u0bae\u0bb0\u0bc1\u0ba4\u0bcd\u0ba4\u0bc1\u0bb5\u0ba4\u0bcd \u0ba4\u0bc1\u0bb1\u0bc8, \u0bb9\u0bbe\u0bb8\u0bcd\u0baa\u0bcd\u0bb0\u0bcb \u0b95\u0bc1\u0bb4\u0ba8\u0bcd\u0ba4\u0bc8\u0b95\u0bb3\u0bcd \u0bae\u0bb0\u0bc1\u0ba4\u0bcd\u0ba4\u0bc1\u0bb5\u0bae\u0ba9\u0bc8, \u0baa\u0bbf\u0bb0\u0bbe\u0bb5\u0bbf\u0b9f\u0ba9\u0bcd\u0bb8\u0bcd, RI \u0bae\u0baf\u0b95\u0bcd\u0b95 \u0bae\u0bb0\u0bc1\u0ba8\u0bcd\u0ba4\u0bc1 \u0ba4\u0bc1\u0bb1\u0bc8, \u0b95\u0bc1\u0bb4\u0ba8\u0bcd\u0ba4\u0bc8\u0b95\u0bb3\u0bcd 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title=\"mailto:lgordon@progeriaresearch.org\">lgordon@progeriaresearch.org<\/a><\/p>\n<p>[\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=&#8221;1_2&#8243; saved_specialty_column_type=&#8221;3_4&#8243; _builder_version=&#8221;4.16&#8243; custom_padding=&#8221;|||20px&#8221; custom_padding_tablet=&#8221;20px|||0px&#8221; custom_padding_phone=&#8221;&#8221; custom_padding_last_edited=&#8221;on|phone&#8221; global_colors_info=&#8221;{}&#8221; custom_padding__hover=&#8221;|||&#8221;][et_pb_text _builder_version=&#8221;4.27.2&#8243; custom_padding=&#8221;||0px|||&#8221; custom_padding_last_edited=&#8221;off|desktop&#8221; global_colors_info=&#8221;{}&#8221;]<\/p>\n<h6><span>\u0bb5\u0bc6\u0ba3\u0bcd\u0b9f\u0bbf \u0ba8\u0bcb\u0bb0\u0bbf\u0bb8\u0bcd<\/span><\/h6>\n<div><span>PRF \u0b9a\u0bc6\u0bb2\u0bcd \u0bae\u0bb1\u0bcd\u0bb1\u0bc1\u0bae\u0bcd \u0ba4\u0bbf\u0b9a\u0bc1 \u0bb5\u0b99\u0bcd\u0b95\u0bbf<br \/>\u0ba4\u0bca\u0bb2\u0bc8\u0baa\u0bc7\u0b9a\u0bbf: 401-274-1122 x 48063<br \/><a href=\"mailto:wnorris@brownhealth.org\" title=\"mailto:wnorris@brownhealth.org\" data-outlook-id=\"6ae643af-3829-46f7-8b6a-2ef767034802\">wnorris@brownhealth.org<\/a><br \/><\/span><\/div>\n<p>[\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 module_class=\u201dfooter\u201d _builder_version.0\u201d4. background_color=\u201d#29327a\u201d custom_margin=\u201d-2px|||||\u201d custom_padding=\u201d0|0px|0|0px|false|false\u201d z_index_tablet=\u201d500\u2033 border_width_top=\u201d12px\u201d border_color_top=\u201d#00b2e2\u2033 global_module=\u201d133\u2033 \u0baa\u0bc2\u0b9f\u0bcd\u0b9f\u0baa\u0bcd\u0baa\u0b9f\u0bcd\u0b9f\u0ba4\u0bc1=\u201d global_colors_info=\u201d{}\u201d][et_pb_row_inner column_structure=\u201d1_3,1_3,1_3\u2033 disabled_on=\u201don|on|on\u201d admin_label=\u201dRow\u201d _builder_version=\u201d4.21.0\u2033 _module_preset\u201d 2dheult_preset\u201d 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use_theme_fonts=\"yes\"][vc_column_text]<strong>Protocol for Culturing Transformed Lymphocytes<\/strong>\r\n\r\nFrom Shipment of Frozen Cells\r\n\r\nFrozen cells will be received in 1 ml aliquots on dry ice. Keep cells frozen until thawed for cell culture or place in liquid nitrogen storage if not starting cultures right away. However, it is recommended that if you are going to store the cells for later use, grow up fresh stocks and freeze multiple ampoules. The cells are cryopreserved in 45% RPMI-1640, 50% fetal calf serum, and 5% DMSO (tissue culture grade).\r\n<ol>\r\n \t<li>Place 10 ml of RPMI-1640, 15% fetal calf serum (FCS), \u00b1antibiotics in a T-25 flask.<\/li>\r\n \t<li>Allow the media to equilibrate in a 37\u00b0C humidified incubator with 5% CO 2in air for 30 minutes.<\/li>\r\n \t<li>Thaw each ampoule of frozen cells in a 37\u00b0C water bath or a beaker of lukewarm water, one at a time.<\/li>\r\n \t<li>Quickly clean the outside of the ampoule off with a sterile isopropanol prep pad, then wrap the prep pad around the ampoule to protect fingers and crack the seal on the ampoule inside a biological safety hood.<\/li>\r\n \t<li>Remove the 1 ml of frozen cells with a sterile glass Pasteur pipet and place in a T-25 flask with 10 ml of medium. Clearly label flask to identify each cell line.<\/li>\r\n \t<li>Place the cells in a 37\u00b0C humidified incubator with 5% CO 2in the air.<\/li>\r\n \t<li>After 24 hours remove 5 ml of medium from the top without disturbing lymphocytes settled on the bottom of the flask and replace with 5 ml of pre-warmed medium.<\/li>\r\n \t<li>Feed the cells with 5 - 6 ml of fresh medium every 3 - 4 days and split into new cultures as required.<\/li>\r\n<\/ol>\r\n<strong>From Shipment of Live Cultures<\/strong>\r\n<ol>\r\n \t<li>Live lymphocyte cultures will be received in T-25 flasks filled to the top with RPMI-1640, 15% FCS, 1% Penicillin-Streptomycin (Gibco). The caps will be tight and sealed with parafilm.<\/li>\r\n \t<li>Remove parafilm.<\/li>\r\n \t<li>Place cultures in a 37\u00b0C humidified incubator with 5% CO2 in air and allow cells to settle out (20 - 30 minutes).<\/li>\r\n \t<li>Using a sterile pipet remove all but 10 - 15 ml of medium and discard.<\/li>\r\n \t<li>Place caps back on flasks loosened slightly to allow gas exchange (Check to make sure cap threads do not have media on them. If so you may want to transfer to a new flask.) Place the cultures back in the incubator with 5% CO 2in air. They should recover in a day or two.<\/li>\r\n \t<li>Feed the cells with fresh medium every 3 - 4 days by resuspending the cell aggregates and removing 50 -60% of the volume and replacing with fresh media. Cells removed from the flask can be used to initiate new cultures as needed.<\/li>\r\n<\/ol>\r\n<strong>For Further Information Please Contact:<\/strong>\r\n\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\u00a0Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\u00a0Department of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA\u00a0Medical Director, The Progeria Research Foundation\r\n\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\n<a href=\"mailto:Leslie_Gordon@brown.edu\">Leslie_Gordon@brown.edu<\/a>\r\n<div><span style=\"font-family: Georgia, Palatino;\">Wendy Norris<\/span><\/div>\r\n<div><span style=\"font-family: Georgia, Palatino;\">PRF Cell and Tissue Bank\r\nPhone: 401-274-1122 x 48063\r\n<a href=\"mailto:wnorris@lifespan.org\" target=\"_blank\" rel=\"noopener noreferrer\">wnorris@lifespan.org<\/a><\/span><\/div>\r\n[\/vc_column_text][\/vc_column][\/vc_row]\t\t","_et_gb_content_width":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-1004","page","type-page","status-publish","hentry"],"yoast_head":"<!-- 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