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cell separation

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cell separation

Postby tregis6 » Fri Jun 20, 2008 6:17 pm

during the separation of the peripheral blood mononuclear cell, i' over-estimated the number of the lymphocytes. Cells having been put at the -80°C during 72H, then in nitrogenize liquid, i would like to know if they don't risk to die during the thawing :cry: :cry: :cry:
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Postby biohazard » Mon Jun 23, 2008 6:40 am

In what kind of medium you froze them, in what volume, how many cells you estimated there was, and how many cells in excess do you think you have?

Generally speaking, you can successfully freeze (and subsequently thaw) anything between 0,5 to 20 million lymphocytes in 1 ml of medium supplemented e.g. with AB serum and DMSO, so I believe you should be okay unless you stuffed like 100 M cells into your cryovial ;)

And naturally, some cells always die during thawing, the number depending on how successful the freezing and thawing steps are.
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Postby tregis6 » Tue Jun 24, 2008 12:03 pm

the composition of medium is for 50 ml: 43,5 ml RPMI + 5 ml FBS + O,5 ml MEM + o,5 ml L-GLUTAMINE + O,5 ml PENICILLIN-STREPTOMYCIN.
The number of cells was over-estimated by a factor of 5. After the counting,we divide the number of cells by 20 million for knowing the size of medium and DMSO use to freeze 20 million lymphocytes per cryovial.We supplement the medium with DMSO 20%.; for e.g for 1 ml of medium we supplement 1 ml of DMSO 20%.So i fear for the volume of DMSO which is also overstimated.
Could you give me a good protocol to minimize cells dying during the thawing.
Thanks for helps
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Postby biohazard » Tue Jun 24, 2008 6:38 pm

As described in Allergy Methods and Protocols (ed. by Jones & Lymphany):

1. Take the cryovial to laminar flow hood and disinfect the outside of the vial.
2. Thaw the vial in glowed hands by slowly swirling it, or quickly thaw in +37C water bath.
3. Open the vial and transfer the cells to a 15 ml Falcon tube with 12 ml of +4C medium in it.
4. Centrifuge the cells for 5 min in 500 g at RT, then discard the supernatant, add fresh RPMI, and repeat this washing procedure as above twice. The cells are then ready to use.
(You may also bring the cells to -20C freezer from the nitrogen tank to warm before the actual thawing)

The protocol I use, works as well:

1. Take the vial from liquid nitrogen and thaw it in +37C water bath (takes approx. 2 mins for 1 ml)
2. Transfer the cell+freezing medium suspension into a 15 ml Falcon tube
3. Pipette slowly, drop by drop, ~3,5 ml of RT or +4C RPMI on the cells.
4. Pipette additional ~3,5 ml of RPMI on the cells, stir/mix gently. This can be done a bit faster.
5. Centrifuge the cells for 8 mins in ~1200 rpm (dunno what the exact g is here, use whatever is standard spin in your lab) and discard the supernatant.
6. Add fresh RPMI (with AB serum for longer storage / growth). You can also repeat the washing step if you feel like it, I rarely do.

You can also thaw several vials in one 15 or 50 ml Falcon tube, just scale up the amount of RPMI accordingly.

The general idea is to dilute and remove the DMSO, but not to do it too fast because the osmotic shock will lyse the cells.

Hope this helps!
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