至尊木虫 (知名作家)
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【答案】应助回帖
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感谢参与，应助指数 +1
suqingru(西门吹雪170代发): 金币+3, 鼓励回帖交流 2014-07-28 11:13:39
这类方法在染色之前或在染色之时都有细胞固定的过程，所以你不需要担心细胞脱离的问题。下面是具体的protocol：
The optimal conditions for monitoring cytotoxicity are to have the cells in the log phase of growth and not to exceed 106cells/cm2. We recommend that each test is performed in a final volume of 200μl and includes a 200μl control sample of cell free medium to be used to blank absorbance readings.
NOTE: We recommend performing tests in serum‐free media to reduce background where possible.
1. Following treatment with appropriate test agents, remove the 96‐well plate to a sterile tissue culture hood and gently layer 50μl Fixative Reagent onto each well.
NOTE: During and after the Fixative Reagent step, the plates should be disturbed as little as possible. Sudden shaking or jolting could dislodge cells and result in inaccuracies in protein quantity.
2. Incubate the plate for 1 hour at 4°C.
3. Wash the wells 3‐4 times with water and vigorously flick the plates between washes to remove excess water. The wash removes excess fixative and serum proteins.
4. Air dry the plates overnight if storage is required or incubate in a 45‐50°C incubator for 30 minutes to remove excess wash.
5. Add 100μl SRB Dye Solution to cover the culture surface of the wells and incubate for 30 minutes at room temperature in the dark.
6. Repeat the wash step (Step 3) using 1X Dye Wash Solution instead of water.
7. Air‐dry the plate as in Step 4.
NOTE: Once the SRB Dye Solution has been added the plates should be protected from light.
8. Add 200μl SRB Solubilization Buffer to each well. Mix by pipetting up & down or gentle agitation to dissolve the dye completely.
9. Measure the absorbance at 550‐580nm, 565nm is the absorption maximum, with a microplate reader.
NOTE: If intense color is visualized then utilize a suboptimal wavelength (490‐530nm) to bring the readings within the linear instrumental range.
CALCULATION OF RESULTS
Calculate the percent cytotoxicity for a given experimental treatment, by using the average absorbance values from experimental, cell control. Ensure that absorbances are blanked with the cell free medium controls.
% Cytotoxicity= (100 x (Cell Control – Experimental)) ÷ (Cell Control) |
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