f*****s
发帖数: 104 | 1 From a screening, we found a small molecule that can inhibit the activity of
a transcription factor. We used MTT assay to show that the small molecule
can inhibit the growth of a tumor cell line which depends on the abnormal
activation of the transcription factor. However, since the TF controls many
metabolism pathways, a reviewer asks to use a different type of assay.
I know nothing about this field (a collaborator did this part). Promega and
Invitrogen list so many kits.
I would like to ask for any suggestions.
Thanks! |
s******r
发帖数: 2876 | 2 直接数细胞。
From a screening, we found a small molecule that can inhibit the activity of
a transcription factor. We used MTT assay to show that the small molecule
can inhibit the growth of a tumor cell line which depends on the abnormal
activation of the transcription factor. However, since the TF controls many
metabolism pathways, a reviewer asks to use a different type of assay.
I know nothing about this field (a collaborator did this part). Promega and
Invitrogen list so many kits.
I would like to ask for any suggestions.
Thanks!
【在 f*****s 的大作中提到】
: From a screening, we found a small molecule that can inhibit the activity of
: a transcription factor. We used MTT assay to show that the small molecule
: can inhibit the growth of a tumor cell line which depends on the abnormal
: activation of the transcription factor. However, since the TF controls many
: metabolism pathways, a reviewer asks to use a different type of assay.
: I know nothing about this field (a collaborator did this part). Promega and
: Invitrogen list so many kits.
: I would like to ask for any suggestions.
: Thanks!
|
d****7
发帖数: 109 | |
m***i
发帖数: 2013 | |
h********n
发帖数: 4079 | |
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
【在 f*****s 的大作中提到】
: From a screening, we found a small molecule that can inhibit the activity of
: a transcription factor. We used MTT assay to show that the small molecule
: can inhibit the growth of a tumor cell line which depends on the abnormal
: activation of the transcription factor. However, since the TF controls many
: metabolism pathways, a reviewer asks to use a different type of assay.
: I know nothing about this field (a collaborator did this part). Promega and
: Invitrogen list so many kits.
: I would like to ask for any suggestions.
: Thanks!
|
c******6
发帖数: 928 | 7 C12 - Resazurin viability assay.
O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (
resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity
. European Journal of Biochemistry. 2000;267:5421-6. |
x*****7
发帖数: 58 | 8 Promega has a kit that can be used for this purpose. It can be done in 96
well plate, but you should have a machine which can read the fluorescent. |
x*****7
发帖数: 58 | 9 Promega has a kit that can be used for this purpose. It can be done in 96
well plate, but you should have a machine which can read the fluorescent. |
h*******o
发帖数: 4884 | 10 Resazurin assay is usually based on NADH/NADPH, not best applicable for
compounds that interfere with mitochondrial function.
cytotoxicity
【在 c******6 的大作中提到】
: C12 - Resazurin viability assay.
: O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (
: resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity
: . European Journal of Biochemistry. 2000;267:5421-6.
|
|
|
A******y
发帖数: 2041 | 11 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. |
h*******o
发帖数: 4884 | 12 3-NPA does that. 3-NPA directly inhibits succinate dehydrogenase activity.
At sub-toxic doses cells are still viable, but SDH activity could be significantly inhibited.
MTT/MTS/Calcein Am/Resazurin assays are often over-used or somewhat used
incorrectly to measure cell viability/cell number.
your
seen
【在 A******y 的大作中提到】
: 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.
|
r***e
发帖数: 2539 | 13 MTT/MTS/Calcein Am/Resazurin assays are often over-used or somewhat used
能展开说说吗？谢谢！ |
j******n
发帖数: 941 | 14 SRB assay, very easy to conduct. |
h*******o
发帖数: 4884 | 15 As mentioned above, a lot of the cytotoxicity/cell viability assays
are based on mitochondrial function, particularly SDH activity. This renders
a lot of the assays under question when the "manipulation/treatment" have
potential impacts on mitochondrial function.
For cancer cell lines, it is less of an issue because of the
notorious "warburg" effect through which cancer cells are more glycolytic
and rely much less on mitochondrial OXPHOS.
But for primary cultures, like neurons, it could be a big issue.
【在 r***e 的大作中提到】
: MTT/MTS/Calcein Am/Resazurin assays are often over-used or somewhat used
: 能展开说说吗？谢谢！
|
r***e
发帖数: 2539 | 16 学习了，谢谢！
renders
have potential impacts on mitochondrial function.
【在 h*******o 的大作中提到】
: As mentioned above, a lot of the cytotoxicity/cell viability assays
: are based on mitochondrial function, particularly SDH activity. This renders
: a lot of the assays under question when the "manipulation/treatment" have
: potential impacts on mitochondrial function.
:
: For cancer cell lines, it is less of an issue because of the
: notorious "warburg" effect through which cancer cells are more glycolytic
: and rely much less on mitochondrial OXPHOS.
: But for primary cultures, like neurons, it could be a big issue.
:
|
