I am working on THP1-XBlue cells to evaluate the inhibition of the NF-kB pathway by drugs and LPS.
From what I have understood, the exposure to LPS would cause the THP1 cells to differentiate into macrophages and adhere to the walls of the well in a 96-well plate.
Hence, do I need to centrifuge and take the supernatant for the SEAP colorimetric assay? What about the control wells where the cells are not exposed to LPS (in this case the cells remain in suspension)? Also, how would centrifuging before taking the supernatant affect the results of my assay?
A gentle centrifugation at 300xg (in a standard microfuge 2500-3000 rpm) for 2 min at 4 degree C and collection of cell supernatant carefully will allow measurement of SEAP activity without interference by proteins that may be released due to cell lysis (due to centrifugation at high g value).
There are certain centrifuges that allow you to spin down the cells in microplates in swing-buckets. If you cannot find some of those, it's is okay. Possible way of doing the assays: have a incubation volume of 120-150 uL per well in your microplate. At the end of the treatment period, take 50 uL out of the original incubation plate with a multichannel pipette, and transfer the liquid (the "pseudo-supernatants") to a complete new plate pre-loaded with the color development agent (50-100 uL per well; or as per instruction). This way, you don't necessarily have to do the contrifugation, which is practically hard to achieve working with microplates.
I would say yes, you should centrifuge samples before supernatants collection as suggested by Divaker Choubey. I would suggest a little bit slower centrifuge (max 2000 rpm in standard microfuge).
Thank you for your suggestions! I will take them into account for my next assay.
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