Hello,
I am trying to isolate cell membrane from a human cancer cell line. At the end of the procedure, I am not sure whether I got the cell membrane. BCA assay did not show any color. Below is the procedure that I followed. Please point out any mistakes and suggest the best practices.
1)A549 cells were grown in T-175 culture flasks to full confluency (50 million cells) in RPMI medium with 10% FBS.
2)Further, cells were detached using EDTA-based enzyme-free cell dissociation buffer (https://www.thermofisher.com/order/catalog/product/13151014) and washed in PBS three times by centrifuging at 1000g for 5min.
3)The cell pellet was dispersed in 2 mL of hypotonic lysing buffer consisting of 20 mM Tris-HCl pH = 7.5, 10 mM KCl , 2 mM MgCl2, and 1 EDTA-free mini protease inhibitor into a single cell suspension and then subjected to 50 passes using disposable homogenizer (https://www.polysciences.com/default/biomasher-iisupsup-disposable-micro-tissue-homogenizer-non-sterile).
4)The cell suspension was centrifuged at 3,200 × g for 5 min. Pellet was discarded. The supernatant was centrifuged at 20,000 × g for 20 min, after which the pellet was discarded and the supernatant was centrifuged again at 100,000 × g for 1 hour. The pellet that should contain the plasma membrane material was then washed once in 10 mM Tris-HCl pH = 7.5 and 1 mM EDTA. The final pellet was collected and characterized for the cancer cell membrane.
Questions:
1) At the end of the procedure (4th step), I did not see any visible pellet after centrifuging at 100,000g. Should we actually see a visible pellet at the bottom to confirm the isolation of cell membrane?
2) Is the step 3 in the protocol OK? Do I need to soak the cell pellet in hypotonic solution for a while? Does the cell pellet present in the hypotonic solution needs to be broken down using the homogenizer? (I broke the cell pellet using forward and reverse pipetting) Is the disposable homogenizer I used good enough?
3) Do you see any loopholes in the protocol? Can you suggest any improvements/best practices for isolation of cell membrane?
Thank you very much for your kind help
The 1st thing I would worry about is whether the homogenization technique is sufficient to break the cell membranes down to the level of small vesicles, because that it what you are relying on during the subsequent differential centrifugation steps. If the membrane particles are too large, they will be in the pellet rather than the supernatant during one or more of the pre-100,000 g centrifugation steps.
Try using a high-power microscope to look for cell fragments after homogenization. You should not see any significant amount.
Thank you very much Adam for this important point and your suggestion. I will follow this.
Do you think anything differently I need to do the homogenization step?
Your homogenizers may be OK, but you should check to make sure.
I used to use sonication for CHO cells, but with quite a large sample. It's tricky to use it with small samples reproducibly A bead beater is another option.
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