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cell loss upon centrifugatino - (Jul/14/2009 )

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i am isolating synovial cells from synovial tissue...
i am finding problems with centrifugation as am lossing the cells after each wash. i am using the same protocol for washing and centrifugation that i use on ligament and cartilage cells and it works perfectelly ....
but not with synovial ....

anybody knows anything special about synovial... or any idea about problem of cell loss upon centrifugation
thanks

-abohollolo-

abohollolo on Jul 14 2009, 03:39 AM said:

i am isolating synovial cells from synovial tissue...
i am finding problems with centrifugation as am lossing the cells after each wash. i am using the same protocol for washing and centrifugation that i use on ligament and cartilage cells and it works perfectelly ....
but not with synovial ....

anybody knows anything special about synovial... or any idea about problem of cell loss upon centrifugation
thanks

I have no experience with synovial cells, but if you think these cells have lesser density than the other cells, try centrifuging at higher rcf or for longer time. You can check out this way.

Do your usual spin. Take out the sup and rather than throwing it away, spin harder and see if you see any pellet. If you do, than change your protocol to higher rcf.

-cellcounter-

what kind of synoviocytes are you isolating? fibroblast- or macrophage-like cells?
the fibroblast like synoviocytes can be centrifuged at speed as low as 150g for 5 to 10 minutes, depending on the volume of cells to centrifuge.
what is your protocol?

-little mouse-

little mouse on Jul 14 2009, 07:06 AM said:

what kind of synoviocytes are you isolating? fibroblast- or macrophage-like cells?
the fibroblast like synoviocytes can be centrifuged at speed as low as 150g for 5 to 10 minutes, depending on the volume of cells to centrifuge.
what is your protocol?



I dont know if my protocol specifies any cell type... i am doing short digestion of synovial tissue for one hour with high collagenase concentration of 0.3% . (because of the research hypothesis, we want to perform this fast digestion). and after digestion just collect and wash and cenrtifuge at 1500 rpm (44g) in 50 ml falcon tube for 5 min . (i get around 1.3 g of tissue ) ....

i wounder if using smaller tubes will help ?

-abohollolo-

I was using 0.1% for 3 hours.
centrifuged 10 min 1200 rpm 4C
washed with medium
centrifuged again 10 min and put the pellet in a 25 cm2 flask

Is it really 44g for 1500rpm? If yes it might be too low. I don't know your rotor, but with mine, 1200 rpm was 130g

-little mouse-

I was using 50 mL tubes too

-little mouse-

A physical loss of cells or a loss of viability?

-GeorgeWolff-

GeorgeWolff on Jul 21 2009, 04:05 AM said:

A physical loss of cells or a loss of viability?


sorry it is 440 g

the loss is physical .. the pellet and cell numer get less through centrifugation

how much tissue weight do you use ? i get everytime about 2 g of tissue...

by the way i would like also to check with you the tissue source if you allow me.
i use rabbits . and get synovial tissue from the the internal side of rabbits knee fat pad. i am trying 3 rabbits each time and getting about 2 g.

so i think i am getting small amount of tissue . recently i used 15 ml tubes and got better pellet preservation.

-abohollolo-

I had human samples. I never weight them.
I don't know how to help you. Do you filter the cells? (you should not)
I didn't. I put the pieces of tissue (after digestion and wash) in a culture flask and wait for the cells to grow outside of the tissue.

-little mouse-

little mouse on Aug 2 2009, 11:53 PM said:

I had human samples. I never weight them.
I don't know how to help you. Do you filter the cells? (you should not)
I didn't. I put the pieces of tissue (after digestion and wash) in a culture flask and wait for the cells to grow outside of the tissue.



Why shouldnt i filter the cell?
i filter for chondrocytes and fibroblasts and is working well

anyway. i used smaller tubes (15 ml) and is giving better results.

thanks for the answers

-abohollolo-
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