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Some opinions needed.


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2 replies to this topic

#1 Psyclick

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Posted 11 February 2010 - 08:19 AM

Hi all

As an intro, this place has been my favourite place to lurk around when i have problems especially in cell cultures. A lot of things i can still learn from all.

Noticed some issues which i need help from the experienced people, so Thanks In advance.
By the way i tried searching around with no results

1. Regarding DMSO for cell freezing, the previous lab i saw buys small glass aliquots of DMSO (5mls) which ensure the freshness of the chemical. Now i am working from a big bottle of DMSO instead, so wondering will the "decrease in freshness" harm the cells i am freezing soon. To be fair, the bottle don't get opened too often.

2. Different cell lines have different recommended media formulations, but I had seen some labs using eg. DMEM/F12 + L-glut + Sodiumbicarb + 10%FBS for all different cell lines which are not recommended by ATCC. Wondering if anyone can comment on this situation, and if there is really a need to remedy this situation.

3. Our lab's SOP for cell freezing is always with 10% DMSO, while ATCC recommends 5%. Wondering if there is any big difference, and which one should i stick with.

THanks again for all's help, hope i will be experienced enough in the future to contribute here too. :lol:

#2 rhombus

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Posted 11 February 2010 - 08:47 AM

Hi all

As an intro, this place has been my favourite place to lurk around when i have problems especially in cell cultures. A lot of things i can still learn from all.

Noticed some issues which i need help from the experienced people, so Thanks In advance.
By the way i tried searching around with no results

1. Regarding DMSO for cell freezing, the previous lab i saw buys small glass aliquots of DMSO (5mls) which ensure the freshness of the chemical. Now i am working from a big bottle of DMSO instead, so wondering will the "decrease in freshness" harm the cells i am freezing soon. To be fair, the bottle don't get opened too often.

2. Different cell lines have different recommended media formulations, but I had seen some labs using eg. DMEM/F12 + L-glut + Sodiumbicarb + 10%FBS for all different cell lines which are not recommended by ATCC. Wondering if anyone can comment on this situation, and if there is really a need to remedy this situation.

3. Our lab's SOP for cell freezing is always with 10% DMSO, while ATCC recommends 5%. Wondering if there is any big difference, and which one should i stick with.

THanks again for all's help, hope i will be experienced enough in the future to contribute here too. :lol:



Dear Psyclick,

Some answers:-

1. I have used the same 500ml bottle of "tissue culture grade" DMSO for the last 10 years.......no problems. I do however only open it in the TC cabinet, and only keep it open for the short time it takes to pipette the required volume. It is then locked away so that only I can use it.

2. In my experience in cell culture (approx 30 odd years), we have regularly used different media than is recommended by the ATCC. Sometimes this is because of experimental protocol but most times it is because we have alot of DMEM or RPMI, and do not have Waymouths, Hams F10/F12 etc.
It is very rare that a cell line will not propagate in another media. We find that the source of FCS is more important than media.

3. The DMSO concentration used in the cryogenic preservation of cell lines is generally 10%. Some cell lines differentiate in DMSO so you should use 10-20% Glycerol as your cryopreservative. Some cell lines are particularly affected by DMSO, as DMSO is cytotoxic to cells. 5% DMSO is therefore used in these circumstances. However cell viability is reduced......this is not normally a problem as cells initiated from frozen is used as a "Seed" i.e. as long as there is enough viable cells, the culture will proliferate to confluence.


Hope this is useful.

Kindest regards.

Uncle Rhombus,

#3 pito

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Posted 11 February 2010 - 09:16 AM

Not having the same amount of experience as rhombus, I can only agree with what he says.

Buying small amount of DMSO is very expencive and not needed if your work is decent.

But as rhombus said: keep your bottle somewhere that others know its yours so no one else can use your bottle or better: abuse your bottle.

If you do not want to open the big bottle to many times then just make some "working bottles" lets say a few tubes with 15 ml DMSO in that you can use then during that year or whatever time you want.

Edited by pito, 11 February 2010 - 09:16 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





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