Suspension Cell Culture, Removal of Dead Cells - (Mar/19/2007 )
Hi there,
Can anyone help me please. I've recently been sent a B cell lymphoma cell line which is a suspension culture, and usually I culture adherent cultures. My problem is this, during the postage of these cells the viability went down quite significantly, so can anyone help me with a technique to actually remove the dead cells? Many thanks.
-shiggins-
Hallo,
I have heard thatfor adherent cells it could be better ACD solution instead of EDTA to supply.
Is it true?
I don't know exactly, but we can listen the next replys
greetings
-creed-
QUOTE (shiggins @ Mar 19 2007, 09:01 AM)
Hi there,
Can anyone help me please. I've recently been sent a B cell lymphoma cell line which is a suspension culture, and usually I culture adherent cultures. My problem is this, during the postage of these cells the viability went down quite significantly, so can anyone help me with a technique to actually remove the dead cells? Many thanks.
Can anyone help me please. I've recently been sent a B cell lymphoma cell line which is a suspension culture, and usually I culture adherent cultures. My problem is this, during the postage of these cells the viability went down quite significantly, so can anyone help me with a technique to actually remove the dead cells? Many thanks.
Dead Cells are generally lighter than live cells. What I have done in the past is to resuspend the cells in a Falcon centrifuge tubes and allow the cells to settle. After 2-3 minutes (depending upon cell type), most of the live cells will have sedimented to the bottom, leaving a mixture of Live/Dead cells floating in the supernatant. Carefully take off supernatant and discard. This will leave the live cells ready for resuspension into the culture flask. You will of course lose some live cells in this process, but as long as you have not exceeded the split ratio, you will be fine.
-Rhombus-
Many thanks, I will give this a try
-shiggins-
QUOTE (Rhombus @ Mar 19 2007, 09:49 AM)
QUOTE (shiggins @ Mar 19 2007, 09:01 AM)
Hi there,
Can anyone help me please. I've recently been sent a B cell lymphoma cell line which is a suspension culture, and usually I culture adherent cultures. My problem is this, during the postage of these cells the viability went down quite significantly, so can anyone help me with a technique to actually remove the dead cells? Many thanks.
Can anyone help me please. I've recently been sent a B cell lymphoma cell line which is a suspension culture, and usually I culture adherent cultures. My problem is this, during the postage of these cells the viability went down quite significantly, so can anyone help me with a technique to actually remove the dead cells? Many thanks.
Dead Cells are generally lighter than live cells. What I have done in the past is to resuspend the cells in a Falcon centrifuge tubes and allow the cells to settle. After 2-3 minutes (depending upon cell type), most of the live cells will have sedimented to the bottom, leaving a mixture of Live/Dead cells floating in the supernatant. Carefully take off supernatant and discard. This will leave the live cells ready for resuspension into the culture flask. You will of course lose some live cells in this process, but as long as you have not exceeded the split ratio, you will be fine.
We normally give a low speed spin to remove the dead cells - It is assumed that spinning down at low speed like ~850-900rpm for 5` would enable only the live cells to settle down while most dead cells will float - same principle as above - but this does not remove all dead cells, we'll still have lot of dead cells in the culture. Well, the V% will go up. I didn't try the above decantation method though.
-padma_dp-