1. Preparation of Lung ECM:

• Obtain fresh mouse lung tissue and rinse with PBS to remove blood or debris by perfusing 3 ml PBS through the trachea or main bronchi.
• Place the lung tissue on a cell strainer 70 microns on a 50 ml falcon tube.
• Gently mash them with a plunger to extract the cells and tissue fragments.
• Rinse the cell strainer with sterile PBS to collect any remaining tissue pieces.
  A Pro tip: Lift the cell strainer a bit from time to time to improve PBS drainage.
• Centrifuge the collected cells at 2000 RPM for 7 minutes to remove red blood cells.
• Implement red blood cell lysis if needed (Sigma Product No. R 7757):

• Add 1 ml of buffer to the cell pellet.
  • Gently mix for 1 minute.
  • Dilute the buffer with 15 ml of PBS.
  • Centrifuge at 2000 RPM for 7 minutes and decant the supernatant.

• Resuspend the cell pellet in full media (DMEM, 10% FCS, 0.5 % Pen-Strep, 1% glutamine, 1% NEAA, 1% pyruvate,1% fungizone) at a density of 200,000 cells per 75 microliters with 37.5 microliters of supernatant from HL-60 cells that are enriched with growth factors.
• Plate the lung cells onto a 96-well plate and incubate for 72 hours to give time for cells to attach and secrete the ECM.

• Preparation of Lung ECM:

• After 72 hours of incubation, remove the media from each well.
• Wash each well with 100 microliters of sterile PBS to remove any cellular debris.
• Place 100 microliters of sterile distilled water (DDW) into each well to decellularize the wells for 30 minutes.
• Remove the DDW and add 37.5 microliters of fresh ______ full media to each well. (depending on the cell line)

• Seeding of Cells:

• Prepare a cell suspension of 20,000 cells in 37.5 microliters of ____ full media. (depending on the cell line)
• Plate the cell suspension in quadruplicates on a 96-well plate containing either lung-decellularized ECM or plastic.
• Switch  ________ full media to serum-free media with or without treatments after overnight incubation. (depending on the cell line)

• Incubate the plate at 37°C and 5% CO2 for ____ hours. Recommendation (48-72 hours)

• Analysis:

• After the 96-hour incubation period, assess the cell viability and proliferation using standard assays such as CCK-8, and collect supernatants.
• Compare the results of the cells cultured on lung decellularized ECM with those cultured on plastic to determine any differences in cell behavior.

For 96-well plates, you can download a protocol template: