Hi,
I am trying to embed cells in low melting point 4% water agarose gel but have been loosing cells due to residual sodium phosphate buffer left in the centrifuge tube. My procedure is below:
1) Fix cells in 5% glutaraldehyde
2) I wash my fixed cells in sodium phosphate buffer for 15min and spin them at 2500 rpm to form a pellet. -I do this 2 times
3) I then melt the agarose and use a warm glass pasture pipette which I place at the bottom of the centrifuge tube and release the agarose to distribute among cells.
4) I centrifuge the tube at 2500rpm to reform the pellet.
5) I place into a 2-8°C fridge to allow the agarose to fully gel usually 30min to an hour.
6) I cut the gel out of the centrifuge tube with razor blade
When I cut the tube and attempt to pull the cells out some of the cells are embedded in the gel but much of the pellet is still liquid. I think that the leftover buffer is to blame but I am not sure how I can remove all of it. Could I use a small triangle of filter paper between step 2 and 3? Maybe add water wash step and spin before the agarose? Would this cause problems with later processing such as osmium or dehydration steps? Not sure what else I could do.
Any advice would be greatly appreciated!
I routinely do this with no problems. Any buffer left after the spinning just dilutes the agarose a bit. It all hardens nicely. It may be it the agarose you are using ???
My procedure;
0.2 g Agarose, Type VII (Sigma A9045) was added to 5 ml distilled water and heated until dissolved.
The agarose was stored in the freezer. 10 seconds and then 15 seconds in the microwave melts it nicely for immediate use straight from the freezer.
The samples are pelleted in 1.5 ml eppendorf tubes and the supernatant removed. Clean plastic pasteur pipets are used to transfer a small amount of the agarose into the eppendorf tube. The agarose and sample were gently mixed and then left for the agarose to solidify (usually in the fridge). After solidifying, the pellets are cut out and removed from the tubes and placed into room temperature buffer and then further processed.
I forgot to add the spinning step after adding the agarose to the cells; to reform the pellet in my procedure; just as you do. It all sets.
Cheers
Hi Brent,
Thank you for the reply. I have been using Promega LMP Agarose (Cat PRV2111 through Fisher). I will try mixing the material and agar more with the pipette and see if that helps. The only thing i worry about is the material solidifying before it can be spun into a proper pellet lowering the cell density per section. I may try out the sigma agarose.
Thank you again!
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