I am trying to scale up a transwell migration assay for PBMCs and I have preliminary data from 96 well plate format, that shows that my cells migrate towards my attractant through a 5 µm polycarbonate membrane.
No I am trying to establish the same in the 24 well format and am having issues with this. With the same chemoattractant concentration, I see less than 30% of my migratory effect in the 24 well format when compared to the 96 well plate effect I observed so frequently.
The TC-treated inserts I use in the 24 well plate also have 5 µm pores, however membranes are made from PET (polyethylene terephthalate).
Could it be that my cells stick to the PET membrane more and fail to migrate to the lower compartment? Are there any other ways the membrane material could impact migration of PBMCs in this setup?
Hello Sofya Novikova
The membrane material could impact the migration of cells. Choosing the optimal membrane type will depend on the type of experiment. As you know that there are three types of membrane material such as polycarbonate (PC), polyethylene terephthalate (PET), and polytetrafluoroethylene (PTFE) each having their own strengths and weaknesses.
1. PC membranes used in inserts are normally treated to promote cell attachment and are made to have high pore density to allow more exchange of cell culture media through the membrane. Thus, PC inserts are best suited for migration studies.
2. PET membranes with lower pore density allow greater transparency for microscopy and imaging. Low pore density PET, therefore, is the membrane of choice when microscopic examination and/ or imaging is necessary. However, there are studies that have used PET for migration assays.
3. Finally, PTFE is also highly transparent for microscopy and has low fluorescence background for immunofluorescence studies. However, the low binding properties of the PTFE material require coating with extracellular matrix proteins prior to seeding to enable the cells to properly attach.
So, you may choose according to the requirement of your experiment (whether you need to observe the cell state or whether you need a membrane with higher pore density). I feel you should continue using polycarbonate membrane inserts as it works better than PET membrane inserts for your experiment.
Best.
Dear Malcolm Nobre,
thank you dearly for your reply. This was very helpful. I will order 24 well polycarbonate membranes next chance I get. Wish me luck!
Best,
Sofya
Dear Sofya,
I wish you Good Luck for your experiments!
Regards,
Malcolm
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