请教用MTT方法测过细胞proliferation的高手们

sannia

第一次做这个实验，有几个问题想请教各位：
+ w5 @1 R% H7 R, B1. 用96孔板做实验，一般每个样品要测几个孔，还是一整版一个样品？# m, x* ^& ?! M, o- O; [6 W
2. 加SDS-HCl 时要把贴壁细胞也吹起来吗？还有这一步的37度温育时间一般多少小时为好？! j* M- t/ E' Y4 S7 f% L0 i4 o7 A7 v7 I
3. 测出来的吸光值怎么对应到细胞个数，自己要先做个直线先？4 m; b6 Y1 f! @8 C

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多谢多谢' @0 l* }* V5 l6 G  _8 {* ~

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- \. }% w3 x( G我用的试剂盒是
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, d# N6 b. m, D3 V0 S1 BVybrant® MTT Cell Proliferation Assay Kit& x4 [, U/ K- A+ j% W
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Experimental Protocol 是：
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Reagent Preparation) n/ r- Z+ K) {# V& O6 Q: B( ?. \

, A8 G; Q$ ^* T1 v    Prepare a 12 mM MTT stock solution by adding 1 mL of sterile PBS to one 5 mg vial of MTT (Component A). Mix by vortexing or sonication until dissolved. Occasionally there may be some particulate material that will not dissolve; this can be removed by filtration or centrifugation.  Each 5 mg vial of MTT provides sufficient reagent for 100 tests, using 10 µL of the stock solution per well. Once prepared, the MTT solution can be stored for four weeks at 4°C protected from light.
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  e, H' ?1 o+ t6 @  d0 A; |    Add 10 mL of 0.01 M HCl to one tube containing 1 gm of SDS (Component B).  Mix the solution gently by inversion or sonication until the SDS dissolves. Once prepared, the solution should be used promptly. Each tube makes sufficient solution for 100 tests, using 100 µL per well.
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Culturing Cells# W% N2 x- a+ L9 P7 d/ D. h# `
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The culture conditions used to grow the cells can affect the results and must be taken into consideration when analyzing the data. The age of the cultures, number of passages and details of the growth medium can all be important factors. Natural variation in the requirements and growth rates of different cell lines make it difficult to provide precise guidelines for preparing your cells. In general, cells seeded at densities between 5000-–10,000 cells per well should reach optimal population densities within 48-–72 hours. Note that the presence of phenol red in the final assay samples can seriously affect results. We strongly recommend that the cells be cultured in medium free of phenol red, if possible. Alternatively, the final incubation with the MTT can be performed after exchanging the cells into medium free of phenol red.
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  ^6 B. y. M# v6 O, U, I' pLabeling Cells8 ^5 x6 }/ j) l/ \
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    For adherent cells, remove the medium and replace it with 100 µL of fresh culture medium. For non-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 µL of fresh medium.3 J& |. C8 O- Q, [/ q$ I  q

) q, J' I- u. U8 O( I& K    Add 10 µL of the 12 mM MTT stock solution (prepared in step 1.1) to each well. Include a negative control of 10 µL of the MTT stock solution added to 100 µL of medium alone.
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1 X' [, E1 @8 R    Incubate at 37°C for 4 hours. At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.
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    Add 100 µL of the SDS-HCl solution (prepared in step 1.2) to each well and mix thoroughly using the pipette.% e! G8 o: Z' ?2 U, s4 y) l

0 t2 Z4 g- d& N# K% J3 \* X    Incubate the microplate at 37°C for 4– hours in a humidified chamber. Longer incubations will decrease the sensitivity of the assay.9
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    Mix each sample again using a pipette and read absorbance at 570 nm.  T9 t( l# ]* ^2 B1 A( F

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Quick Protocol Option/ [2 x: P+ y1 o7 s6 P% W" @

4 X5 t0 i7 H' F1 P/ V! M+ mTo shorten the time of the assay it is possible to use DMSO (not provided) as a solubilizing agent to dissolve the formazan.67 m9 @; {, B; l' a' f7 r/ t" }( H
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    After labeling the cells with MTT, as described above, remove all but 25 µL of medium from the wells. For non-adherent cells it may be necessary to first centrifuge the plates to sediment the cells.( h+ G- o* T; D* q) g
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    Add 50 µL of DMSO to each well and mix thoroughly with the pipette./ y- C, ]% e9 r! H  K) \, O

" L, U. J' n6 |$ N    Incubate at 37°C for 10 minutes.
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0 G. p8 D/ v6 Z, r3 M8 Q: i    Mix each sample again and read absorbance at 540 nm not 570 nm, as above.

sannia

回复 sfk06 的帖子
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多谢多谢！
7 [) @& j- }$ ^  M加SDS-HCl后一般孵育多少小时呢？
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sannia

回复 forcat 的帖子
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  Y& S/ x& p) L6 m解释非常详尽，感激不尽！

sannia

多谢提醒！回头做了再来汇报结果。