Problem with primary cortical culures from P1 mice - (Jul/18/2008 )
Hello,
Lately we've been encountering a problem with our primary neuron cultures. We're using cortical tissue from P1 C57B/6 wildtype mice. Basically we dissect the tissue in HBSS, put it in HBSS in a 15mL non-cytogenic tube, spin 1000*g 2 minutes, remove HBSS, add 5mL HBSS with trypsin (no EDTA, 2.5 mg/mL trypsin, 0.85 mg/mL NaCl) and resuspend the pellet by pipetting. We incubate at room temperature 15 minutes, then spin at 1000*g for 5 min. The problem is that we're not getting a nice cell pellet, instead it looks like it is barely attached to the bottom and then there is this gelatinous, filamentous, fern-like goo semi-attached to the bottom of the tube and waving in the supernatent. If we try to remove the goo from the top it will come out all in one piece and detach itself from the bottom of the tube.
I was reading that some people keep the tissue in small pieces and let them settle rather than centrifuging, and only separate the tissue using a pipette after trypsin treatment rather than before. Would this be what is causing the gelatinous product and lack of cell pellet? I am positive that it is not contamination, as it only happens after adding the trypsin and I can see the solution of cells getting thicker as it digests.
On another note, I've also experienced this type of non-pelleting, frond-like residue when I did a miniprep one time a few months ago, and no trypsin was involved then...
Thanks for your help!
- Kelley.
Lately we've been encountering a problem with our primary neuron cultures. We're using cortical tissue from P1 C57B/6 wildtype mice. Basically we dissect the tissue in HBSS, put it in HBSS in a 15mL non-cytogenic tube, spin 1000*g 2 minutes, remove HBSS, add 5mL HBSS with trypsin (no EDTA, 2.5 mg/mL trypsin, 0.85 mg/mL NaCl) and resuspend the pellet by pipetting. We incubate at room temperature 15 minutes, then spin at 1000*g for 5 min. The problem is that we're not getting a nice cell pellet, instead it looks like it is barely attached to the bottom and then there is this gelatinous, filamentous, fern-like goo semi-attached to the bottom of the tube and waving in the supernatent. If we try to remove the goo from the top it will come out all in one piece and detach itself from the bottom of the tube.
I was reading that some people keep the tissue in small pieces and let them settle rather than centrifuging, and only separate the tissue using a pipette after trypsin treatment rather than before. Would this be what is causing the gelatinous product and lack of cell pellet? I am positive that it is not contamination, as it only happens after adding the trypsin and I can see the solution of cells getting thicker as it digests.
On another note, I've also experienced this type of non-pelleting, frond-like residue when I did a miniprep one time a few months ago, and no trypsin was involved then...
Thanks for your help!
- Kelley.
After you trypsinize the tissue pieces, you have to add DNase to disassociate them, else they will be one piece of gooey or jelly like stuff. The sticky stuff is nucleic acid and even after miniprep thats what is released.
Hope this helps.