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Getting cells from flasks


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#1 epigenetics

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Posted 04 August 2009 - 11:42 AM

Hello,

I am going to start CHIP assay for histone acetylation study at our lab. We got Millipore Magna CHIP kit. According to this one and also in other protocols, i saw they are getting cells by scraping. We grow our cells in flasks and then do trypsinization (0.125%) to get the cells. When i talked to Millipore Tech support, they said, "first do crosslinking and then trysinization. but it is also true that after crosslinking , the cell are very resistant to trypsin. So it may take 15 minutes, even after that also you might need to scrape the cells off from petridish".

Provided that we are using flasks for growing cells and trypsin to get cells off, I am really confused here. Is there any body who is using trypsin instead of scrapper and working on CHIP assay? would you please share how are you doing it.
Considering the number of samples we have, it is impossible to use scrapper for each of the samples for us.
please advise me.
Thanks,

#2 orlatron

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Posted 04 August 2009 - 05:09 PM

Hello,

I am going to start CHIP assay for histone acetylation study at our lab. We got Millipore Magna CHIP kit. According to this one and also in other protocols, i saw they are getting cells by scraping. We grow our cells in flasks and then do trypsinization (0.125%) to get the cells. When i talked to Millipore Tech support, they said, "first do crosslinking and then trysinization. but it is also true that after crosslinking , the cell are very resistant to trypsin. So it may take 15 minutes, even after that also you might need to scrape the cells off from petridish".

Provided that we are using flasks for growing cells and trypsin to get cells off, I am really confused here. Is there any body who is using trypsin instead of scrapper and working on CHIP assay? would you please share how are you doing it.
Considering the number of samples we have, it is impossible to use scrapper for each of the samples for us.
please advise me.
Thanks,


I do ChIP on adherent primary cells. I use the Farnham lab protocol, not a kit. I frequently trypsinise 10 T175 flasks at a time for ChIP...Nonetheless, my protocol is:

Wash flask 2 twice in 1x PBS
0.25% trypsin + EDTA until cells detached, neutralise with 10% serum centrifuge and count
Resuspend 10e7 cells in 50mL medium
Add 37% formaldehyde to 1% v/v, 8min on rocker at RT
Add glycine to 0.125M concentration 5min at RT, then store on ice
450xg 5min at 4 degrees, then 2x wash in ice cold PBS at 4 degrees
Snap freeze/continue with ChIP

This seems to work fine - the cells are definitely cross linked, ChIP works...Hope that is helpful. :lol:

#3 epigenetics

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Posted 05 August 2009 - 06:14 AM

Thanks a lot for the info. It is really helpful to know. So you are doing crosslinking in 50 ml propelyne tube and that works. Good to know.
Just one thought, as you are doing trypsinization first and then crosslinking, dont you think that trypsinization is killing your adherent proteins and it might hurt other protein DNA interactions, and might not reflect whats happening invivo.
It just came to my mind as i dont know about this a lot, please let me know your thought.

thanks,


Hello,

I am going to start CHIP assay for histone acetylation study at our lab. We got Millipore Magna CHIP kit. According to this one and also in other protocols, i saw they are getting cells by scraping. We grow our cells in flasks and then do trypsinization (0.125%) to get the cells. When i talked to Millipore Tech support, they said, "first do crosslinking and then trysinization. but it is also true that after crosslinking , the cell are very resistant to trypsin. So it may take 15 minutes, even after that also you might need to scrape the cells off from petridish".

Provided that we are using flasks for growing cells and trypsin to get cells off, I am really confused here. Is there any body who is using trypsin instead of scrapper and working on CHIP assay? would you please share how are you doing it.
Considering the number of samples we have, it is impossible to use scrapper for each of the samples for us.
please advise me.
Thanks,


I do ChIP on adherent primary cells. I use the Farnham lab protocol, not a kit. I frequently trypsinise 10 T175 flasks at a time for ChIP...Nonetheless, my protocol is:

Wash flask 2 twice in 1x PBS
0.25% trypsin + EDTA until cells detached, neutralise with 10% serum centrifuge and count
Resuspend 10e7 cells in 50mL medium
Add 37% formaldehyde to 1% v/v, 8min on rocker at RT
Add glycine to 0.125M concentration 5min at RT, then store on ice
450xg 5min at 4 degrees, then 2x wash in ice cold PBS at 4 degrees
Snap freeze/continue with ChIP

This seems to work fine - the cells are definitely cross linked, ChIP works...Hope that is helpful. :)



#4 cellcounter

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Posted 05 August 2009 - 06:36 AM

Hello,

I am going to start CHIP assay for histone acetylation study at our lab. We got Millipore Magna CHIP kit. According to this one and also in other protocols, i saw they are getting cells by scraping. We grow our cells in flasks and then do trypsinization (0.125%) to get the cells. When i talked to Millipore Tech support, they said, "first do crosslinking and then trysinization. but it is also true that after crosslinking , the cell are very resistant to trypsin. So it may take 15 minutes, even after that also you might need to scrape the cells off from petridish".

Provided that we are using flasks for growing cells and trypsin to get cells off, I am really confused here. Is there any body who is using trypsin instead of scrapper and working on CHIP assay? would you please share how are you doing it.
Considering the number of samples we have, it is impossible to use scrapper for each of the samples for us.
please advise me.
Thanks,

I don't trypsinize before or after formaldehyde treatment. I just crosslink, block, wash in PBS, scrape the cells in PBS with protease inhibitors, spin them down and add lysis buffer to the pellet. Works wonderfully on many adherent lines I have worked on.

#5 KPDE

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Posted 05 August 2009 - 09:50 AM

Thanks a lot for the info. It is really helpful to know. So you are doing crosslinking in 50 ml propelyne tube and that works. Good to know.
Just one thought, as you are doing trypsinization first and then crosslinking, dont you think that trypsinization is killing your adherent proteins and it might hurt other protein DNA interactions, and might not reflect whats happening invivo.
It just came to my mind as i dont know about this a lot, please let me know your thought.

thanks,


Hello,

I am going to start CHIP assay for histone acetylation study at our lab. We got Millipore Magna CHIP kit. According to this one and also in other protocols, i saw they are getting cells by scraping. We grow our cells in flasks and then do trypsinization (0.125%) to get the cells. When i talked to Millipore Tech support, they said, "first do crosslinking and then trysinization. but it is also true that after crosslinking , the cell are very resistant to trypsin. So it may take 15 minutes, even after that also you might need to scrape the cells off from petridish".

Provided that we are using flasks for growing cells and trypsin to get cells off, I am really confused here. Is there any body who is using trypsin instead of scrapper and working on CHIP assay? would you please share how are you doing it.
Considering the number of samples we have, it is impossible to use scrapper for each of the samples for us.
please advise me.
Thanks,


I do ChIP on adherent primary cells. I use the Farnham lab protocol, not a kit. I frequently trypsinise 10 T175 flasks at a time for ChIP...Nonetheless, my protocol is:

Wash flask 2 twice in 1x PBS
0.25% trypsin + EDTA until cells detached, neutralise with 10% serum centrifuge and count
Resuspend 10e7 cells in 50mL medium
Add 37% formaldehyde to 1% v/v, 8min on rocker at RT
Add glycine to 0.125M concentration 5min at RT, then store on ice
450xg 5min at 4 degrees, then 2x wash in ice cold PBS at 4 degrees
Snap freeze/continue with ChIP

This seems to work fine - the cells are definitely cross linked, ChIP works...Hope that is helpful. :)


I doubt the trypsin will get into the cells and digest any chromatin proteins but if you are looking for an interaction which is rapidly turned over and may be dependent on cytoskeletal organization or adherence of the cells then that interaction might be lost. Is there any way you can grow the cells in plates instead of flasks and scrape them instead of trypsinize? This is ideal since you can fix them immediately with formaldehyde without disrupting them before they're fixed.

#6 little mouse

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Posted 05 August 2009 - 11:20 PM

I never did Chip assay, but I think the best choice is to fix the cells with formaldehyde and then to scrap. Once fixed, you won't trigger any signaling pathway by trypsinization or suspending your cells.
You can scrap your cells in a flask, choose the right cell scraper. i already did it , not a big problem.




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