Strategy to detect cell survival - for primary cells that can not be trypsinized (Dec/27/2006 )
Hello, everyone,
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
-open2654-
QUOTE (open2654 @ Dec 28 2006, 06:17 AM)
Hello, everyone,
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
for direct inspection use trypan blue; often used is MTT/MTS assay, also SRB assay; numerous assays for necrosis and apoptosis are available;
try to detach cells by only EDTA w/o trypsin; scraping off is another alternative
-The Bearer-
Have you tried to use trypsin inhibitor to stop the enzymatic activity?
or you can do the vitality assay on cells with different numbers than normalize your assay with total protein or nucleic acid contents.
-genehunter-1-
QUOTE (genehunter-1 @ Dec 28 2006, 08:39 AM)
Have you tried to use trypsin inhibitor to stop the enzymatic activity?
or you can do the vitality assay on cells with different numbers than normalize your assay with total protein or nucleic acid contents.
or you can do the vitality assay on cells with different numbers than normalize your assay with total protein or nucleic acid contents.
Hi, Hunter
Yes, I neutralize trypsin by serum containing medium and this procedure works well with wield type cells.
If I want to normalize the assay with total protein, I have to seed another backup plate for the protein assay, am I right? Because I used MTT for cell viability, I can not use the exact same well for protein assay.
By the way how to detect nucleic acid contents as you mentioned?
Thanks!
-open2654-
QUOTE (kosmodrom @ Dec 28 2006, 06:22 AM)
QUOTE (open2654 @ Dec 28 2006, 06:17 AM)
Hello, everyone,
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
for direct inspection use trypan blue; often used is MTT/MTS assay, also SRB assay; numerous assays for necrosis and apoptosis are available;
try to detach cells by only EDTA w/o trypsin; scraping off is another alternative
Hi,
Thank you for the reply. What is the concentration of EDTA used to detach cells? And what is the inhibitor for EDTA after cells are detached?
Thanks
-open2654-
Good point. MTT will interfere with both assays. How about if you use live-and-death staining method? (Invitrogen/Molecular Probes)
-genehunter-1-
QUOTE (kosmodrom @ Dec 28 2006, 03:22 PM)
QUOTE (open2654 @ Dec 28 2006, 06:17 AM)
Hello, everyone,
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
I have a problem in detecting survival of primary cultured cells.
Those cells are fibroblast delivered from new born SOD knockout mice kidney. They can grow in the media. My purpose is to compare their survival after different reagent treatments. To do that, I have to trypsinize them and reseed them at the same cell numbers for different treatments. However, after trypsinization, they are almost all dead. As I understand it is a nature of this KO cells, i.e. they are hardly stand trypsinization. So, anyone has idea in this case how to detect the cell survival?
Thanks a lot.
for direct inspection use trypan blue; often used is MTT/MTS assay, also SRB assay; numerous assays for necrosis and apoptosis are available;
try to detach cells by only EDTA w/o trypsin; scraping off is another alternative
they may be differences in effective EDTA conc between different cell lines; try to start with the commonly used conc of your trypsin/EDTA solution; as EDTA is non-toxic (beside pH effects) you can double the conc
-The Bearer-