鐢�(ch菐n)鍝佷粙绱�
绱拌優鍚嶇ū锛�鎱㈡€х矑绱拌優鐧借鐥呯窗鑳�
褰㈡厠(t脿i)鐗规€э細鎴愭穻宸寸窗鑳�
鐢熼暦鐗规€э細鎳告诞鐢熼暦
鐗瑰緛鐗规€э細瑭茬窗鑳為仼鍚堝仛杞�(zhu菐n)鏌撳涓伙紝渚嗚嚜浜庝竴浣�53姝插コ鎬ф參鎬х矑绱拌優鐧借鐥呬汉鐨勮兏鑵旂⿳娑�
鍩归(y菐ng)姊濅欢锛� IMDM锛�
Iscove锛噑 Modiefied Dulbecco锛噑 Medium 10%FBS
鍌充唬鏂规硶锛� 1锛�
3鍌充唬,2-3澶╁偝涓€浠�
鍌充唬鎯呮硜锛� PN5
鍑嶅瓨姊濅欢锛�鍩虹(ch菙)鍩归(y菐ng)鍩�+5%DMSO+20%FBS
鐢�(ch菐n)鍝佸悕绋� | 鎱㈡€х矑绱拌優鐧借鐥呯窗鑳� |
鑻辨枃绨$ū | K-562 |
鐙€鎱�(t脿i) | 璨煎 |
绱拌優鍐峰噸淇濆瓨锛�
1.鍑嶅瓨娑诧細92%瀹屽叏鍩归(y菐ng)鍩�+8%DMSO锛堝彲浠ユ牴鎿�(j霉)瀵﹂瀹ゆ浠惰嚜琛岄伕鎿囷級
2.闄嶆韩姝ラ锛�4搴�10min锛�-20搴�2h锛�-80閬庡鍚庢恫姘繚瀛�銆�
绱拌優鍩归(y菐ng)鐨勬搷浣滄椹燂細
1.鎱㈡€х矑绱拌優鐧借鐥呯窗鑳�鍚歌蛋鐢ㄤ簬鍩归(y菐ng)鍩猴紝鍔犲叆3-5mL PBS鍚庤紩杓曟檭鍕曞煿椁�(y菐ng)鐡舵饯娲楃窗鑳�锛�
2.鍚稿共鍑圥BS鍚庡姞鍏�1mL 0.25%鑳伴叾-0.53mM EDTA锛岃紩杓曟悥鏅冨煿椁�(y菐ng)鐨夸娇鑳伴叾娴告矑绱拌優琛ㄩ潰锛屽煿椁�(y菐ng)鐡舵斁37搴﹀煿椁�(y菐ng)绠辨秷鍖�锛�
锛堢窗鑳炲皪浜庢秷鍖栨瘮杓冩晱鎰�锛屾秷鍖栭亷搴︽渻鍤撮噸褰遍熆绱拌優鐙€鎱�(t脿i)锛屽皫(d菐o)鑷寸窗鑳炲偝浠f浜℃紓娴垨鑰呭偝浠e悗涓嶉暦銆傜窗鑳炴秷鍖栧埌绱拌優闁撻殭璁婂ぇ浣嗘湭鑴惤锛屽彲浠ヨ紩杓曞惞涓嬫檪鍗冲彲绲傛锛岀姝㈡秷鍖栧埌绱拌優瀹屽叏婕傛诞?锛涢澒铓f瘞楹渞鐩¢噺杓曟煍锛屼笉瑕佸惞鍑哄ぇ閲忔埃娉�銆傦級
3.鍔犲叆6-8ml瀹屽叏鍩归(y菐ng)鍩虹祩姝㈡秷鍖栵紝杓曡紩鍚逛笅绱拌優娣峰嫽锛�
4.灏囨贩鍕荤殑绱拌優1000rpm锛堢磩150g锛夐洟蹇�3min锛屾涓婃竻锛屽啀鐢ㄦ柊楫煿椁�(y菐ng)鍩洪噸鎳�37搴﹀煿椁�(y菐ng)绠辩辜绾�(x霉)鍩归(y菐ng)锛�
鍌充唬姣斾緥: 涓嶅悓绱拌優鐢熼暦閫熷害涓嶄竴锛屽叿楂斿偝浠f瘮渚嬭绱拌優鐢熼暦閫熷害鑰屽畾锛屽ぇ閮ㄥ垎绱拌優閬╃敤1:3-1:4鍌充唬锛岀敓闀疯純鎱㈢殑绱拌優鍙互1:2鍌充唬銆�