3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

1. 3D co-cultures with direct interaction

1. Label BrC cells and monocytes with different fluorescent dyes before cell culture to allow independent sorting of each cell lineage after culture for analysis of mRNA and protein expression. Use commercially available derivatives of coumarin and rhodamine that freely pass through the cell membrane of living cells. A general protocol for labeling is the following:
   1. Prepare a monolayer of BrC cells of interest (2 × 10⁶ cells in a 25 cm² culture flask) and replace the standard medium with sufficient pre-warmed working solution of the fluorescent dye (1:2,000 of the fluorescent dye in standard base medium without any supplement). Incubate 30 min at 37 °C in a humidified 5% CO₂ environment. Aspirate the dye solution and rinse gently with sufficient 1x PBS. Aspirate the PBS and add the standard medium.
   2. Trypsinize the cell monolayer with 2 mL of a solution of 0.05% trypsin and 0.48 mM EDTA for 5-10 min at 37 °C. Add 200 µL of FBS to stop trypsinization and 7 mL of the correspondent medium without supplements. Resuspend the cells by pipetting. Take an aliquot of 10 µL to count cells in a Neubauer chamber. Continue with the co-culture protocol after cell counting.
   3. Pellet the cells at 430 x g for 5 min at RT (perform this first since monocytes grow in suspension). Discard supernatant and gently resuspend cells with 3 mL of a pre-warmed working solution of the fluorescent dye (1:2,000 of the fluorescent dye in a standard base medium without supplements). Incubate for 30 min at 37 °C in a humidified 5% CO₂ environment.
   4. Pellet the cells again, discard the dye working solution, and gently resuspend with 5-7 mL of 1x PBS. Pellet once more, discard the PBS, and resuspend cells in 5-7 mL of standard medium. Take an aliquot of 10 µL of cell suspension to count cells in a Neubauer chamber. Continue with the co-culture protocol after cell counting.
2. Set the co-culture as follows: spread enough ECME at the bottom of the well to form an even layer in each well of a 4-well chamber slide system. Plate 20 µL of a single cell suspension containing 5 × 10⁵ labeled BrC cells per well.
3. After 15-20 min of incubation at 37 °C, add a suspension of 2.5 x 10⁵ labeled monocytes in 80 µL assay medium (supplemented with 60% ECME).
4. Allow ECME to solidify for 15-20 min at 37 °C.
5. Add 1 mL of a 1:1 mix of the BrC cells and monocyte culture media.
6. Incubate co-cultures at 37 °C in a humidified 5% CO₂ environment for 24 h, 48 h, or 5 days to track changes at different time points.
7. Degrade ECM proteins to recover the cells from the cultures. Aspirate and discard the medium, add 0.5 mL of 1x PBS with 0.1% trypsin and 0.25% EDTA, and incubate for 3 h at 37 °C. After incubation, add 0.5 mL of 1x PBS with 10% FBS to neutralize the trypsin, and resuspend the cells by pipetting vigorously to obtain a single-cell suspension.
8. Pellet cells at 765 x g for 5 min at RT, discard the supernatant, and resuspend the cells in 5 mL of 1x PBS with 10% FBS. Repeat this step once.
9. Subject the cell suspension to fluorescence-activated cell sorting (FACS) with the appropriate instrument. Ensure that the final populations are at least 95% pure).
10. After sorting, wash the cells with sterile 1x PBS, pellet (430 x g for 5 min at RT), and resuspend in sterile 1x PBS. Cells can be processed according to specific protocols for RNA isolation or protein analysis.
11. Repeat each assay three times, including 3D cultures of each individual cell lineage as controls.