Primary mouse DRG immunocytochemistry troubleshooting - cell detachment

Another thread about ICC and cells not being adherent enough. We’ve recently had some issues with DRGs not making it through ICC. The edges have all the cells and center has mostly lost them.

Here is an example from a recent chamber well

We do a PDL and Laminin coating before plating. This is what we do:

Plate Coating

• Coat plate wells or coverslips with PDL (0.1mg/ml) overnight. Place in 37C tissue culture incubator.
o For Ibidi 12 well, use 150μl of PDL
• On the day of dissection, aspirate the PDL solution from all wells with a sterile pipette tip attached to a vacuum pump. Rinse the dishes twice with autoclaved ultrapure water by repeating the pipetting and aspiring step. Dry the plates in the biosafety cabinet.
• Fill each well with 150 μL of laminin solution (3 ug/ml). Place the plates in the 5% CO2 incubator at 37ºC for 1–3 h.
• Aspirate laminin and let plate dry in biosafety cabinet.

Any thoughts? @tberta @sshiers @dmolliver

Our protocol looks pretty standard, kind of like this one:

This issue is not all the time, but intermittent. I’m wondering if the laminin might not be as good as it once was. Will try new batches of things. Maybe we need to be even gentler as well with washes after fixing.

Try fixing the cells with 100% ethanol for 30mins after PFA fixation and add 1.2mM calcium chloride to your wash buffer.

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Alex, did you ever try adding CaCl2 to your wash buffer? I’m wondering if this would be helpful. We are struggling with the same issue, especially with our human DRG cell line, but also occasionally with mouse DRG preps. Did you ever find other solutions?

For what it’s worth, we don’t dry our laminin-coated plates before use, we rinse with water and aspirate the water right before use. No idea if this matters.

HI @dmolliver
Thanks for checking in on this.
We figured it out, but can’t be entirely sure what.
but I went back to basics. I got new, fresh laminin from Sigma.
and as you say above, instead of letting the laminin dry, we went right into wells immediately after aspirating, following this protocol Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release - PubMed
It seems to have worked. We had abundant neurons on the glass this time.

So my take away:

  1. Fresh reagents including PDL and laminin
  2. Don’t let laminin dry

Nice, thanks for letting me know!