I've been using LUHMES cells to differentiate into dopaminergic neurons for over 6 months now, but i always have issues when it comes to platting for differentiation. It's always an ON/OFF kind of situation, sometimes they adhere and then begin to differentiate(ideal), other times they don't adhere at all or adhere but maintain a round shape and not the typical shape of LUHMES which renders them from differentiating (the scenario that happens more often) which is quite frustrating. I follow the recommended method for platting:
1- Begin culture in T75 - Day 0
2- begin differentiation - Day 1
3- lift cells and plate them on 96/384 well plate (Day 2) and continue differentiation.
I hemi-feed them every other day once plated.
With 96 well, i tried different cell densities when platting (20, 25, 30, 35k/well), when they are able to differentiate, i noticed that anything above 35k they wouldn't adhere or would cluster quite a lot, which is not ideal as I'm using them for imaging.
Does anyone ever had these reoccurring issues with their LUHMES cells and could shed a light to my problems? I'm running out of ideas what could possibly be happening. Cells are fine and happy while in T75/175 flasks. Could it be the plate? i use PerkinElmer PhenoPlate. Also, for imaging, what cell density does everyone go for when using 96/384? I have also plated the LUHMES on day 3, which worked before, but still had the same issue as Day 2 plating.
Thanks in advance!
Try seeding proliferating LUHMES cells onto 96/384 well plate and continue with differentiation the following day (or the day after depending on the cell density) by chnaging the media. Also, I am assuming that you are thoroughly rinsing off the coating solution which can otherwise affect adherence and viability.
i tried that method, and the issue with this method is when you change the media to begin differentiation the cells tend to lift, so not ideal. I figured out the solution around this. I pre coat the plates with fibronectin/Poly-L-Orn as usual, but the day i plan to plate my cells i coat the plate with matrigel (24hrs-3hrs). This solved my issue.
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