Hi Michael

For DRG neurons 15% BSA cushion gives good results, without the need for a cell strainer.

Another method you could try is to transfer the triturated cell suspension for 5-10 minutes, to a 1 ml syringe held vertically with a luer lock so that the cells settle down and the debris floats on the surface; the cells can then be easily let out and separated from the debris.

Good luck

Uma

Thank you Uma! My primary concern would be that the 15% BSA cushion is even higher than the 1% BSA cushion I'm using which is already giving me reduced yield... At what speed do you centrifuge the tube? I'm using 100g's which I'm concerned may not be enough to pellet the cells after a 6 minutes spin.

I'll give the syringe method a try as well, do you usually just homogenize the solution and suck it up into the syringe, or do you usually do the same thing with layering the cells over a BSA containing solution and let the cells settle through that?

You would need to centrifuge at 200 g for 5 minutes to get a pellet.

Transfer the triturated cell suspension with a pippette into a syringe without a BSA cushion.

Hi Uma, 

As an update, I discovered that my loss of yield was due to the fact that I wasn't adequately separating my cells from the undissociated tissue prior to layering it on the BSA cushion. After using a 70 uM cell strainer, the results were significantly better with yield around 2.5 x 10^6 vs 4.5 x 10^6 without the cushion.

I think most of my (remaining) lost yield was from after triturating in the papain, I only centrifuged at 300 gs for 3 minutes. After centrifuging the supernatant at 700gs for 3 minutes I discovered that I left about half of the pellet behind. Do you think I can be a little more aggressive with centrifuging the pellet after trituration?

I'm also noticing that the BSA cushion did not eliminate all debris, there still is a speckling of membrane and on top of that I noticed a significant population of small, white, unattached cells in addition to the (very healthy) looking attached neurons. I noticed this both with and without a 1 hour post adherence media change, with 5% FBS, and 10% FBS recovery. I was *very* gentle with my trituration this time, using a flame pulled 0.5 mm pipette opening with ~1ml/second trituration speed and only pulling from the bottom of the tube I was triturating in to get the undissociated fragments. I did this about 10 times. until most of the solution was homogenous except for a small clump of undissociated cortex.

Any thoughts if the inhibition of the papain might be insufficient? Worthington has only optimized their protocols (according to the scientist in tech support) for 80% recovery which seems rather low...

Best,

Michael

If there is undissociated tissue, you could increase the papain incubation by 10 - 15 minutes.  Aggressive centrifugation will result in non neuronal cells in the pellet.  The TI should be adequate to neutralize the papain, but all solutions should be warm after the incubation.  The exact duration etc will have to be determined by trial and error compared with published protocols on PubMed.

Thanks Uma! I did another p0 culture and increasing it by 5 minutes (20 total) seems to have helped without impacting yield. I used the same inhibition solution (0.1 mg/mL BSA/Ti at 37C for the 6 minute gradient spin down [centrifuge not heated]) as suggested by the worthington protocol.

Also for anyone else who comes across the question, a 1 hour recovery in 5% or 10% FBS before switching to NBM-A/B27/Glutamax does seem to help cell attachment (or maybe prevent cells from dying and detaching). The concentration didn't affect it so I'd go with 5% to minimize serum exposure (which I'm always paranoid about).

Hi Michael

The plated neurons will adhere better if the substrate is coated with poly l or D lysine and laminin (20 micrograms/ml each). For Polylysine coating plate on substrate (coverslips) for 1 hour then aspirate and dry (should be done at least 48 hours in advance). Laminin should be added previous night and incubated in incubator, then aspirated just before plating cells.  The plating medium will require growth factors. see the atttached pdf and link. 

J Vis Exp. 2007; (10): 562.

Published online 2007 Dec 19. doi: 10.3791/562

Hi Uma, 

Thanks for the link. I already coat my coverslips with PDL (no laminin) using a similar protocol (0.2 mg/mL PDL in borate buffer at pH 8.5 for ~6 hours) however have not been using laminin. My media composition is NBM-A/B27/Glutamax, just recovering for 1 hours in NBM-A/B27/Glutamax/5% FBS seems to help cell yield.

Hi Michael,

Can you share the BSA cushion protocol you are using? I know you mentioned some details above but wondering where you found that? I am using the following protocol to culture p0 cells and also having some issues with debris. I am culturing in PLL treated plates and chamber slides: https://www.ncbi.nlm.nih.gov/pubmed/22936216

Thank you!

Rob

Hi Rob,

If you shoot me an email ([email protected]) I will send you a copy of my protocol. I'm using a slight modification of the Worthington Papain Dissociation System.

The biggest hindrance to getting a good cushion is not filtering the cell suspension through a strainer prior to layering . Some people can get similar results letting the chunks settle and pulling off the supernatant post trituration, but I personally have had better yield with the 70 uM nylon strainer.

Using a centrifuge with the ability to slow the acceleration/deceleration is crucial to get good separation of the layers. I use a Beckmann Coulter with the rate of acceleration/decceleration set to the lowest setting at 70x gs for 6 minutes.

Furthermore, plating the cells for 1 hour in 5% FBS and then doing a complete media change 15-60 minutes post plating drastically decreases the amount of debris. 

As an aside, I supplement my dissection buffers with 50 uM APV to prevent excitotoxic cell damage during the procedure.

Using my protocol, I routinely get about 4-5 million cells per pair of cortices from each p0 pup with >95% yield per Trypan blue staining/phase contrast counts. These cultures last about 4-5 weeks on average and I routinely record spontaneous activity from DIV7 onward. I'm sure there is more optimization to be  had (feeding schedule, FUDR/Uridine or AraC for inhibition of glia, composition/osmolarity of culture media and dissecting media, use of physiological media (brainphys), optimization of papain digestion, presence or absence of FBS in plating media, B27 vs Horse serum supplemented media).

Michael