Hi all,
I am using 96-well plates to culture iPSC derived neurons, the problem is, when doing ICC, my neurons tend to detach during the washing procedure, till the final step nearly all neurons are gone. I was already being very careful, and using the speed-control multichannel pipette, still didn’t help. We are using 100µg/mL PLO + 20µg/mL laminine for coating, and neurons never detached in 24-well plates.
I have been troubled by this problem for 2 months…. so it would be of great help if I could hear some advice from you. Do you ever have the problem dealing with neurons in multi-well plate, how you solve it? Also are you using any especial plate?
Thanks in advance!
Due to less surface area, pressure applied during washing may cause detachment of cells.
I never did but learnt that people do neuron culture in not less than 24 well plate (even no manufacturer provide PLO-Laminin coated 96-well plate for Neuron culture).
If you want to save antibody, you should culture your cells on coated coverslips in 24 or 12 well plate. After culture remove the coverslip and fix it on slide to follow the staining protocol.
I am not too sure what kind of 96-well plate you are using, however a glass-bottomed well plate is recommended to use for ICC for better viewing. Another method will be using coated-coverslips, simply put them into wells, then seed your cells on top of the coverslips. By the way, do you use PFA to fix your cells?
Due to less surface area, pressure applied during washing may cause detachment of cells.
I never did but learnt that people do neuron culture in not less than 24 well plate (even no manufacturer provide PLO-Laminin coated 96-well plate for Neuron culture).
If you want to save antibody, you should culture your cells on coated coverslips in 24 or 12 well plate. After culture remove the coverslip and fix it on slide to follow the staining protocol.
Have you tried using PEI? (50% Polyethylenimine Solution (PEI) Sigma-Aldrich P3143). This is what we use at Axion Biosystems for our neuronal assays in multi-well plates and it seems to hold them down well. However, our assays are label free/ reagent free. so there are no washing steps of the neurons to measure the electrical activity.
Jitendra Kumar Shandilya Hi Jitendra, thank you for your answer. Exactly, the mechanic pressure in small area is the reason leading to detachment. I saw in some published papers neurons are successfully stained in 96-well plates, so I am thinking maybe some people have a trick? Also, as I am dealing with 9 cell lines, I prefer multiple well plates, and if it works it actually saves more antibodies.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating