流感診斷試劑盒（快檢方法）

廣州健侖生物科技有限公司

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流感診斷試劑盒（快檢方法）

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【公司名稱】 廣州健侖生物科技有限公司
【市場(chǎng)部】    楊永漢

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【騰訊  】 2042552662
【公司地址】 廣州清華科技園創(chuàng)新基地番禺石樓鎮(zhèn)創(chuàng)啟路63號(hào)二期2幢101-103室

實(shí)驗(yàn)步驟
1．貼壁細(xì)胞
1)   取普通潔凈蓋玻片于70%乙醇中浸泡5分鐘或更長(zhǎng)時(shí)間，無(wú)菌超凈臺(tái)內(nèi)吹干或用細(xì)胞培養(yǎng)PBS或0.9%NaCl等溶液洗滌三遍，再用細(xì)胞培養(yǎng)液洗滌一遍。將蓋玻片置于六孔板內(nèi)，種入細(xì)胞培養(yǎng)過(guò)夜，使約為50%-80%滿。
2)   刺激細(xì)胞發(fā)生凋亡后，吸盡培養(yǎng)液，加入0.5ml固定液，固定10分鐘或更長(zhǎng)時(shí)間（可4℃過(guò)夜）。
3)   去固定液，用PBS或0.9%NaCl洗兩遍，每次3分鐘，吸盡液體。洗滌時(shí)宜用搖床，或手動(dòng)晃動(dòng)數(shù)次。
4)   加入0.5ml Hoechst 33258染色液，染色5分鐘。也宜用搖床，或手動(dòng)晃動(dòng)數(shù)次。
5)   用PBS或0.9%NaCl洗兩遍，每次3分鐘。
6)   滴一滴抗熒光淬滅封片液于載玻片上，蓋上貼有細(xì)胞的蓋玻片，盡量避免氣泡。使細(xì)胞接觸封片液，切勿弄反。
7)   熒光顯微鏡可檢測(cè)到呈藍(lán)色的細(xì)胞核。
2. 懸浮細(xì)胞
1)   離心收集細(xì)胞樣品于1.5ml離心管內(nèi)，加入0.5ml固定液，緩緩懸起細(xì)胞，固定10分鐘或更長(zhǎng)時(shí)間（可4℃過(guò)夜）。
2)   離心去固定液，用PBS或0.9%NaCl洗兩遍，每次3分鐘。洗滌期間手動(dòng)晃動(dòng)。
3)   zui后一次離心后吸去大部分液體保留約50ml液體，再緩緩懸起細(xì)胞，滴加至載玻片上，盡量使細(xì)胞分布均勻。
4)   稍晾干，使細(xì)胞貼在載玻片上不易隨液體流動(dòng)。
5)   均勻滴上0.5ml Hoechst 33258染色液，染色5分鐘。用吸水紙從邊緣吸去液體，微晾干。
6)   用PBS或0.9%NaCl洗兩遍，每次3分鐘。
7)   滴一滴抗熒光淬滅封片液于載玻片上，蓋上一潔凈的蓋玻片，盡量避免氣泡。
8)   H. 熒光顯微鏡可檢測(cè)到呈藍(lán)色的細(xì)胞核。

Experimental steps
1. Adherent cells
1) Take ordinary clean coverslips immersed in 70% ethanol for 5 minutes or longer, blow dry in a sterile clean bench or three times with a cell culture solution such as PBS or 0.9% NaCl and wash with the cell culture solution Again Cover glass slides were placed in six-well plates and seeded in cell culture overnight to make about 50% -80% full.
2) Stimulate the cells to apoptosis, exhaustion of culture medium, add 0.5ml fixative, fixed for 10 minutes or longer (4 ℃ overnight).
3) Remove the fixative and wash twice with PBS or 0.9% NaCl for 3 minutes each to aspirate the fluid. Washing should be shaker, or manually shaking several times.
4) Add 0.5 ml Hoechst 33258 stain and stain for 5 minutes. Also suitable shaker, or manually shaking several times.
5) Wash twice with PBS or 0.9% NaCl for 3 minutes each.
6) Drop a drop of antifluorescence to quench the sealing solution on the slide, cover the cover glass with cells, try to avoid bubbles. Make the cells in contact with the sealing solution and do not turn it off.
7) The fluorescence microscope can detect the blue nucleus.
2. Suspension cells
1) Collect the cell samples by centrifugation in 1.5 ml centrifuge tubes, add 0.5 ml of fixative solution, suspend the cells slowly and fix for 10 minutes or more (4 ℃ overnight).
2) Centrifuge the fixative and wash twice with PBS or 0.9% NaCl for 3 minutes each. Manual shaking during washing.
3) After the last centrifugation to absorb most of the liquid to retain about 50ml liquid, and then slowly suspended cells, added dropwise to the slide, try to make the cells evenly distributed.
4) Slightly dry, the cells attached to the slide is not easy to flow with the liquid.
5) Dilute 0.5 ml Hoechst 33258 stain evenly and stain for 5 minutes. Wipe off the liquid from the edges with blotting paper and allow to dry slightly.
6) Wash twice with PBS or 0.9% NaCl for 3 minutes each.
7) Drop a drop of antifluorescence to quench the sealing solution on the slide, covered with a clean cover glass, try to avoid bubbles.
8) H. Fluorescence microscopy detects blue nuclei.