Probelm with Patch clamp of Dissociated DRG neurons

Hi everyone
We are trying to study the electrophysiological parameters in dissociated DRG primary culture cells, we have been trying for a while now to get a whole cell configuration to do a patch clamp experiments but we were unsuccessful.

the problem seems to be in our cell culture, the cells we are trying to patch do not look good and the is no rigidity in cellular membrane, the whole cell is going inside the pipette upon applying negative pressure.

Does anyone have any ideas how we could get better cell culture to surpass this problem?

Thank you very much in advance

Hi @Abderaouf
Sorry to hear about your problems. Can you be more specific about what exactly is happening? If you can provide some images or traces, you may get more detailed answers from people.

@mny3 @alex_naka @tberta

Hi @achamess
Thanks very much for the reply, as for the details : we are using acutely dissociated DRG neurons, we are taking DRG neurons from the rat , the perform enzymatic digestion using collagenase type IV and trypsin, then we perform mechanical digestion then culture the DRG resulted in petri dishes containing DMEM supplemented with Penicillin+Streptomycin and FBS at 37 C and 5% CO2.
picture are for the results we are getting and bright field microscopy

Hi. Thanks for sharing. Are those even cells? Those round things could be just debris or lipid globules. The cells should be well adhered to the plate. Maybe stain the coverslip with DAPI and do some ICC to confirm. I know you said those are acute culture, but after 1 hr, most should be down on the glass.

Unfortunately i don’t have any stains or anything else to confirm, i Just have bright field microscope, and the cells don’t attach at all to the coverslips.

Are you coating your coverslips with PDL +/- laminin or collagen?

I highly recommend reading the protocol from @dmolliver

Or this one, which is open access

Thank you very much for the reply and the suggested articles, for the coverslips i am using Poly-L-Lysin.