Hello Maria Fernandez

First and foremost, culture HDFs to allow them to deposit their ECM on the surface of the cell culture dish. Then the cells may be removed through decellularization using chemicals or enzymatic digestion, leaving behind the ECM.

Protocol in brief:

1. Seed HDFs in culture vessel of appropriate size.

2. Provide sufficient time for cells in culture (for example 7-8 days) to allow for significant ECM deposition (HDFs naturally secrete ECM components like collagen, fibronectin, and elastin into the surrounding environment).

3. Keep changing the media on a regular basis so that you may have continued ECM production.

4. Once enough ECM is deposited, remove the cells to isolate the ECM.

5. Wash the cells with PBS once.

6. You may use ammonium hydroxide to lyse the cells. Incubate the cells in a solution of PBS with 50 mM NH4OH and 0.05% Triton X-100 for 2 minutes at room temperature and keep observing the cells under a light microscope to watch for cell debris.

7. Wash the ECM 3 times with PBS to remove any remaining cellular debris or decellularization reagents.

8. You may also use DNase I to remove DNA from ECM released from dying cells. Use 20 U/mL solution of DNAse I for 1 hour at 37°C.

9. Wash the ECM 3 times with PBS.

10. The decellularized ECM may be collected from the culture dish and used for SDS- PAGE.

The paper attached below will be useful.

Article Stromagenesis During Tumorigenesis: Characterization of Tumo...

Best,

Hey! So if you wanna get ECM from your HDFs for SDS-PAGE, here’s a simple way to go about it. First, culture your HDFs until they’re nice and confluent (like 90–100%). Once they’ve laid down a good ECM layer, wash the cells gently with PBS a couple of times. Then, to get rid of the cells but keep the ECM, treat them with a buffer like 0.5% Triton X-100 with 20 mM NH4OH in PBS for about 5–10 minutes at room temp—this lyses the cells but leaves the ECM intact. Wash again with PBS gently a few times to make sure all the cell debris is gone. Now, to collect the ECM, you can scrape it off in RIPA buffer or SDS sample buffer (with protease inhibitors, of course), boil it for 5 minutes, spin down the gunk, and then run the supernatant on SDS-PAGE. That should give you a nice ECM protein profile! Let me know if you want to tweak it for specific proteins or downstream stuff.