Hello all,
I have recently embarked into mammalian cell culture using mutiwell culture plates (e.g. 6-well plates, 12-well plates, etc.) as opposed to previous studies where I have used individual flasks (e.g. T25, T75, etc.). When using flasks, I was accustomed to lysing cells in somewhat of a more "low-throughput" method in a lysis buffer containing 1% Triton X-100, applying mechanical shearing force by passing the cells through an 18G needle.
Moving to multiwell culture plates, the needle method is quite tedious. Having to pass the cells from each well through an 18G needle, one at a time, is very time consuming and counterbalances much of the time that is saved by using a multiwell plate in the first place.
Is there another method that is friendlier to "high-throughput" multiwell plates, that anyone might suggest which lyses cells without having to pass them through a needle one-by-one -- yet does not interfere with downstream assays for protein concentration?
Thanks in advance,
Chris Dieni
Dear Dr. Christopher Anthony Dieni
It is not always necessary to pass the cells through an 18G needle.
You may change your lysis buffer. I would recommend the use of RIPA lysis buffer. RIPA extraction buffer contains non-ionic and ionic detergents which can extract protein from a wide variety of cell types (both adherent and suspension cultured mammalian cells), effectively extracting cytoplasmic, nuclear and membrane proteins. RIPA buffer ensures efficient cell lysis and protein solubilization preventing protein degradation and interference with protein immunoreactivity and biological activity. Protein extraction carried out using RIPA is compatible with various downstream applications because most antibodies and protein antigens are not adversely affected by the components of this lysis buffer.
The protocol of cell lysis using RIPA lysis buffer is as follows.
1. Prepare an appropriate volume of ice-cold RIPA lysis buffer. The lysis buffer should be supplemented with protease inhibitors at the time of lysis to minimize protein degradation upon release of cellular proteases. For a 6-well plate add 200-300 µl per well, for a 24-well plate add 100-200 µl per well and for a 96-well plate add 50-100 µl per well.
A. For adherent cells, wash the cells with PBS to get rid of the culture media. Add an appropriate volume of cold RIPA lysis buffer, then mix until the cells immerse completely in the buffer. Shake gently for 5min. Incubate on ice for another 10-15 mins.
B. If you are dealing with suspension cells, centrifuge the cell suspension to collect the cell pellet. Then wash the cells with PBS. Add an appropriate volume of cold RIPA lysis buffer, then mix until the cells are dispersed. Vortex for 5min to fully lyse the cells. Incubate on ice for another 10-15 mins. Please note that all steps of the protein extraction should be performed at 4°C.
2. After the above lysis step, pipette the cell suspension up and down and transfer the cell lysates from the wells to 0.5 mL microcentrifuge tubes.
3. Centrifuge the lysates at 14,000 × g for 5 minutes to remove cell debris.
4. Transfer supernatants to clean tubes. You could either proceed to the next step or store the cell lysates at –20 °C (for 1 month) to perform the assay later.
5. The protein concentration of your lysates may be determined by a total protein assay such as the Bicinchoninic acid assay. You could perform the micro-BCA assay as follows.
You may add 5μL of sample to a 96-well microplate followed by 100μL of BCA reagent. Mix plate thoroughly on a plate shaker for 30 seconds. The plate may be incubated in the dark for 30min at 37 °C. The absorbance may be measured at 560nm, and protein concentration may be determined from a BSA standard curve.
You may prepare RIPA lysis buffer (1X) as follows:
50mM Tris-HCL, pH 7.6
150mM NaCl
1% NP-40 or Triton X-100
0.5% Sodium deoxycholate
0.1% SDS
1mM EDTA
Add protease inhibitor cocktail fresh (1X) or use the following protease inhibitors such as Pepstatin A (1uM), Leupeptin (10uM) and PMSF (0.1mM) when you are about to perform cell lysis. Phosphatase inhibitors will be required to be added in the lysis buffer fresh only when phosphorylation states (activation states) are being investigated. The phosphatase inhibitors include Sodium fluoride (1mM) and Sodium orthovanadate (1mM).
Regards,
Malcolm Nobre
Dear Malcolm
Thank you so much for this recommendation! All of this sounds perfectly logical. If I might ask, for adherent cells, by what manner should I shake gently shake the plate for 5 min?
Thanks again,
Chris Dieni
Dear Dr. Dieni
You may use a plate shaker with a low speed of about 25-50rpm.
Best.
Dear Malcolm Nobre,
Thank you so much for everything!
Best,
Chris
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