k***g
发帖数: 4904 | 1 试了试，细胞太小，显微镜下没法判断是什么颜色啊、
有人能说说不，两三微米大的细菌，能用Trypan Blue测活动性么？ |
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k******y
发帖数: 236 | 2 在贴壁细胞中siRNA了某基因，之后就有大量的碎片漂浮在medium里，镜下看就是小圆
泡，diameter < 10um，而细胞即使trypsin消化下来之后也并不是规则的圆形，这些碎
片并不能uptake trypan blue，计数活细胞(trypan blue-, diameter > 10um) 的数量
有明显减少, 但以trypan blue+ 的％算，并不比对照组减少很多，大概从95％减少到
90％
用FITC-Annexin V和PI double staining，做flow没有看到明显Annexin V+细胞群，
AnnexinV+ PI+细胞群会增加但也不会增加太多(本身％也小于10％)
用50uM Z-VAD-FMK caspase inhibitor 与siRNA共同处理细胞, 这些碎片就不会产生，
计数活细胞的数量也不会减少, 和对照细胞没有差别
请问这是否说明细胞有死亡？能否看出是何种死亡方式？谢谢大家! |
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b******k
发帖数: 1874 | 4 I am writing to ask for help.
I am using Miltenyi anti-NKp46-biotin-bead- MS column kit. The first step is
to get splenocyte from spleen by gentlMACS without enzyme treatment. A very
simple protocol, just put spleen into the PEB buffer and let gentleMACS run
m_spleen_01 program once, and filter, spin, aspirate, resuspend the pellet.
The problem comes from the resuspending process. The small tight pellet will
produce(or contain) big clump that is almost impossible to break when 10 ml
PEB buffer i... 阅读全帖 |
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t**j
发帖数: 10 | 5 Why not stain cells with dyes that detect dead cells? alternatively, the
simplist way is to trypsinize the cells after drug treatment and count cells
in the presence of trypan blue, when dead cells turn blue. Do the same to
the control cells that are the same confluent. |
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s*****g
发帖数: 7857 | 6 cell cycle or apoptosis不行吗?
或直接PI(OR TRYPAN BLUE)染色,数细胞?
of
many
and |
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A******y
发帖数: 2041 | 7 Resazurin, MTT, MTS etc. are all based on mitochondria respiration. If your
cell is attached, I will just do PI stain. You can also count the cell
directly with trypan blue etc.
However, I can tell you the reviewer is been difficult. I bet you already
count the cells and your compounds do inhibits cell growth....I haven't seen
a single compound that could lower the mitochondria function so much that
will affect the MTS assay if it is not directly reflective toward cell
numbers. |
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l***y
发帖数: 4671 | 8 能想到的最可能的解释就是 selective necrosis。
建议做个 mtt 看看。
trypan blue 和 facs 都 assume canonical apoptosis，可能不适用于你的 case。 |
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