I am studying vasculogenic potential of cancer stem cells, and so I culture stem cell spheres on Matrigel. After several days, they form 3D tube structures into the gel.
Inhibition of my protein of interest prevents this tube formation, so I want to interrogate the various angiogenic kinase pathways to see what is being changed between my control and my treated group.
However, phospho-RTK are notoriously unstable and so traditionally Western Blot cell collection has to be done very quickly on ice to preserve the phosphorylation.
What I need is a method to get my cells out of the matrigel (its a soft substrate and they grow INTO it, so I can't just scrape them) so I can lyse them without actually changing them. Accutase (or Trypsin or Versene) won't be effective because a) they can degrade the extracellular RTKs and change their activation and b) it takes too long at 37oC so receptors can lose their phospo groups.
BD has a 4oC cell recovery solution but it takes an hour - could this potentially work?
Thank you for any help you all can provide!
I am not sure about the concerns; I guess using a cell strainer will minimize the chance of gel matrix contamination.
Ultimately, you need the protein from these cells, for western blot.
Why don’t you try to extract protein directly from these 3D cultures?
Please check following link, you can get an idea:
http://www.amsbio.com/brochures/Total_Protein_Extraction_from_Cells_Cultured_in_Alvetex_in_3D_-_AMSBIO.pdf
You can try both the ways, whichever is working well.
Hope this helps
All the very best
My suggestion would be to use syringe plunger.
Break the 3D structure with the help of a syringe, which we usually do to isolate spleen or lymph node cells without enzymes. You can do this process on ice.
Once you can see a homogeneous solution with less clumps, use a cell strainer which will give you a precise single cell suspension.
Hope this can help
All the very best
Interesting idea although I don't actually need single cells - but what is to prevent the gel matrix from passing through the screen and contaminating my protein samples?
Hello Levi.
The companies also sell their own dissociation enzymes and buffer. That is the best to get your cells frozen at the moment of phosphorylation on ice or 4C.
However, if you do not really need single cell suspension, try collagenase for 30 min in your medium without changing the state of treatment with no serum in CO2 incubator , of course. You have GFs only, I guess. Then incubation with gelatinase while you watching the dismantling gel structures.
Regards.
I am not sure about the concerns; I guess using a cell strainer will minimize the chance of gel matrix contamination.
Ultimately, you need the protein from these cells, for western blot.
Why don’t you try to extract protein directly from these 3D cultures?
Please check following link, you can get an idea:
http://www.amsbio.com/brochures/Total_Protein_Extraction_from_Cells_Cultured_in_Alvetex_in_3D_-_AMSBIO.pdf
You can try both the ways, whichever is working well.
Hope this helps
All the very best
Just telling you what we do in our Lab.
Matrigel gellify at 37°C but this process is reversible simply return your plate, with your tube-like structures, to 4°C o.n. The day after applying pre-refrigerated PBS to dilute Matrigel and cells at least 20X and transfer in tubes. Then collect your cells by centrifugation at 400g 5'. After a further wash in PBS, the pellet could be processed for protein extraction.
Hoep this could help somehow
Good luck
I would go for what Simone said, but also if the cold PBS do not work because of low yield, try to do with trypsin. I know people that at the end they found out that trypsin is the best solution to dissolve matrigel. Regarding one reagent that is called BD recovery solution, in my hands and hand from a lot of people did not work so much, just keep in mind that.
Hi Levi,
We use a thermo-responsive hydrogel instead of Matrigel It is liquid at 4'C and stiff at 37'C.. Here is the link: https://www.advancedbiomatrix.com/thermoreversible-hydrogel/mebiol-lyophilized-10-ml-5180-10ml/?gclid=EAIaIQobChMIkKi5t5D51wIVUCOBCh1k0wbmEAAYASAAEgJORPD_BwE
works great, and liquifies pretty fast, it will be the best thing to shorten harvest time.
Warm regards,
Steve
Dear Levi,
We did something similar in the past.
We just snap frozen the matrigel with the cells to prevent dephosphorylation, and a similar drop off matrigel alone as control. Then we grind it in liquid N2 in a mortar and process it in RIPA plus inhibitors (you can keep it in these conditions in ice for a while to improve extraction). The centrifuge the samples a collect the supernatant. I use low volume is of RIPA and a 6X laemmli to avoid further dilution. In my experience I would recommend not to loose time quantifying protein it is not accurate in these conditions. Just load some sample then blot it against housekeeping protein and you can calculate from densitometry the amount s required for an even loading.
Besides, you'll have the sample with the gel alone for control, which we found to not contribute significantly to the phospho profile.
This also worked in assays to detect levels of GTP-bound GTPases which are hydrolyzed extremely fast.
Hope it helps, good luck
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