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Freezing medium and cell counting - (Mar/25/2005 )

Freezing medium:
Should i use ice-cold freezing medium for freezing the cells? At what temperature should it be? blink.gif


Cell counting:
How can i concentrate the original cell suspension if there are too few cells to count? I think i should centrifuge the cells down and then dilute it. Is this okey?



Thanks.

-autumn-

Hi there, well firstly you will have to thaw out freezing media, so put it in a 37 degree water bath until its thawed..then use it....should be fine.

To concentrate the original cell suspension, spin the cells down in the centrifuge and resuspend them in a small amount of media to count them.


Hope that helps smile.gif

-Maria-

Temperature for freezing media: if you are using DMSO and the cell line is a typical lab cell line (immortal/transformed/cancer line), then you can work at room temperature (RT) with the media between RT and 37C. You can leave cells in DMSO containing media for ~ 30 min at RT without viability dropping below 90%. Then freeze at < -70C.

If you are freezing down primary cells or very finicky cells, place the media on ice and work quickly to prevent the cells "drying out" from the anti-freeze agent (DMSO etc.).


Centrifuging before counting is fine. Remember to note the volume you took from the culture and thus centrifuged, and the volume you resuspend the cells. Then you can calculate the density of cells in the original culture.

Hope this helps you smile.gif

-AussieUSA-

QUOTE (Maria @ Mar 29 2005, 08:37 AM)
Hi there, well firstly you will have to thaw out freezing media, so put it in a 37 degree water bath until its thawed..then use it....should be fine.

Thanks, but should i freeze the freezing medium upon storage? unsure.gif

-autumn-

QUOTE (autumn @ Mar 30 2005, 01:31 AM)
QUOTE (Maria @ Mar 29 2005, 08:37 AM)
Hi there, well firstly you will have to thaw out freezing media, so put it in a 37 degree water bath until its thawed..then use it....should be fine.

Thanks, but should i freeze the freezing medium upon storage? unsure.gif



Yup, I would. Some people store their freezing media in the fridge..I think its best to wack it straight in the -20 freezer after use.

I am not sure if you can repeatedly freeze-thaw though.

Hope that helps.

Maria

-Maria-

QUOTE (Maria @ Mar 31 2005, 04:24 AM)
QUOTE (autumn @ Mar 30 2005, 01:31 AM)
QUOTE (Maria @ Mar 29 2005, 08:37 AM)
Hi there, well firstly you will have to thaw out freezing media, so put it in a 37 degree water bath until its thawed..then use it....should be fine.

Thanks, but should i freeze the freezing medium upon storage? unsure.gif



Yup, I would. Some people store their freezing media in the fridge..I think its best to wack it straight in the -20 freezer after use.

I am not sure if you can repeatedly freeze-thaw though.

Hope that helps.

Maria



I'd store the freeze medium in the freezer - and I've never had any trouble with several freeze/thaw cycles up to five or so.

Katherine

-KDHughes-

Dear Autumn,


Not sure what cells you are using or what freezing medium you use but here is my experience with both topics:


1) Freezing medium: I simply make up 90%DMEM and 10%DMSO in a tube and place in water bath until it reaches 37^C. If you use ice cold medium on your warm cells you could cause shock which will damage them. Freeze them down in an special alcohol surrounded container in -80^C, and the container will cause a steady temp decrease of about 1degree a minute. Then after one day put in liquid nitrogen to store as long as you like.

2) When you want to count cells (I assume using haemacytometer, the slides with a cross pattern of squares), centrifuge down and remove all media, then add a small amount of fresh medium (in my case DMEM, 1 ml) and if you mix 20 micro litres of cell mixture to "trypan blue" (shows up dead cells on slide so no need to count them) in ratio 1:1 or whatever you like, you can further adjust concentration.

I.e. a typical haemacytometer has viewing volume of 0.0001ml, so to get concentration per ml multiply by 10000, then multiply by trypan blue dilution factor. If you used equal parts of cells and trypan x2, if twice as much trypan as cells x3 etc.


If you are using DMSO and DMEM then you can keep DMSO in room temp on a shelf, DMEM should be kept in fridge (about 4 degrees C). If you do mix them together before storage then putting both in fridge should be fine. You should not freeze the freezing medium to store it really as the continuous thawing of the DMEM (especially with serum in) may cause problems.

Hope this helps!

-pedrostevo-