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Revival of kasumi-1 cells


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#1 enthusiastic

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Posted 01 April 2011 - 01:07 AM

Hi all
I am facing problems in growing (reviving) Kasumi-1 cell line. The procedure which we follow is- we take stock from liq. N2 then thaw it in water bath at 37 degree for ~2 min. Then we add 1ml of 20% RPMI 1640 media in the cryovial and mix slowly and add it in a centrifuge tube containing 4 ml 20% RPMI 1640 media. We then centrifuge it at 995rpm for 10 mins then discard supernatant and resuspend pellet in 1 ml of 20% RPMI 1640 media and then transfer it into a T-25 flask containing 20% RPMI 1640 media and incubate at 37C, 5% CO2. On revival we observe healthy cells but as incubation time proceeds cell memb starts disrupting and only dead cells are seen and we are unable to get viable cells. :(

Can anybody plz suggest any protocol for revival of Kasumi-1 cells so that I can get healthy proliferating cells for my further work.

Any suggestions will be highly apprciated........

Thanks

#2 Clare

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Posted 04 April 2011 - 12:11 AM

Hello :)

I have used Kasumi cells quite a bit and have had no probs reviving them - perhaps your batch is a bit crap?
They do grow really slowly (I think the doubling time is at least 40 hours) - how long have you had them in culture for?
From memory they are pretty ugly looking cells - how are you confirming they are dead?

Clare

Hi all
I am facing problems in growing (reviving) Kasumi-1 cell line. The procedure which we follow is- we take stock from liq. N2 then thaw it in water bath at 37 degree for ~2 min. Then we add 1ml of 20% RPMI 1640 media in the cryovial and mix slowly and add it in a centrifuge tube containing 4 ml 20% RPMI 1640 media. We then centrifuge it at 995rpm for 10 mins then discard supernatant and resuspend pellet in 1 ml of 20% RPMI 1640 media and then transfer it into a T-25 flask containing 20% RPMI 1640 media and incubate at 37C, 5% CO2. On revival we observe healthy cells but as incubation time proceeds cell memb starts disrupting and only dead cells are seen and we are unable to get viable cells. :(

Can anybody plz suggest any protocol for revival of Kasumi-1 cells so that I can get healthy proliferating cells for my further work.

Any suggestions will be highly apprciated........

Thanks



#3 enthusiastic

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Posted 05 April 2011 - 09:09 PM

Hello

I keep them for minmum of 48 hrs and then feed or change media... The cell line wass cryoprserved on 2007.. Under 40X I observed the disintegrated cell membrane from which I can conclude that cells are dying becoz for few days some cells look healthy and as the incubation continues all the cell dies... :( ..

If you have any protocol plz reply...

Thanx...

Enthusiastic


Hello :)

I have used Kasumi cells quite a bit and have had no probs reviving them - perhaps your batch is a bit crap?
They do grow really slowly (I think the doubling time is at least 40 hours) - how long have you had them in culture for?
From memory they are pretty ugly looking cells - how are you confirming they are dead?

Clare


Hi all
I am facing problems in growing (reviving) Kasumi-1 cell line. The procedure which we follow is- we take stock from liq. N2 then thaw it in water bath at 37 degree for ~2 min. Then we add 1ml of 20% RPMI 1640 media in the cryovial and mix slowly and add it in a centrifuge tube containing 4 ml 20% RPMI 1640 media. We then centrifuge it at 995rpm for 10 mins then discard supernatant and resuspend pellet in 1 ml of 20% RPMI 1640 media and then transfer it into a T-25 flask containing 20% RPMI 1640 media and incubate at 37C, 5% CO2. On revival we observe healthy cells but as incubation time proceeds cell memb starts disrupting and only dead cells are seen and we are unable to get viable cells. :(

Can anybody plz suggest any protocol for revival of Kasumi-1 cells so that I can get healthy proliferating cells for my further work.

Any suggestions will be highly apprciated........

Thanks



#4 Clare

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Posted 07 April 2011 - 02:08 AM

I would suggest keeping the cells in culture for longer and then trying looking at the cells under the microscrope with a dead cell stain like trypan blue. It's such a standard procedure that someone at your place of work should be able to help :)

When I have thawed the cells in the past, I had to grow them for at least 2 weeks in order to get enough live cells for my experiments (around 10^8).

Clare

[quote name='enthusiastic' timestamp='1302066557' post='105883']
Hello

I keep them for minmum of 48 hrs and then feed or change media... The cell line wass cryoprserved on 2007.. Under 40X I observed the disintegrated cell membrane from which I can conclude that cells are dying becoz for few days some cells look healthy and as the incubation continues all the cell dies... :( ..

If you have any protocol plz reply...

Thanx...

Enthusiastic




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