3 replies to this topic

[enthusiastic]

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

[Clare]

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

enthusiastic, on 01 April 2011 - 01:07 AM, said:

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

[enthusiastic]

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

Clare, on 04 April 2011 - 12:11 AM, said:

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

enthusiastic, on 01 April 2011 - 01:07 AM, said:

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

[Clare]

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