4 replies to this topic

[autumn]

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


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.

Edited by autumn, 25 March 2005 - 02:20 PM.

[Maria]

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  

[AussieUSA]

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  

[autumn]

Maria, on Mar 29 2005, 08:37 AM, said:

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?  

[Maria]

autumn, on Mar 30 2005, 01:31 AM, said:

Maria, on Mar 29 2005, 08:37 AM, said:

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?  

<{POST_SNAPBACK}>

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