Fluorescence Based Transwell Macrophage Attraction Assay: An In Vitro Method to Study Tumor-Macrophage Interaction Through Chemical-Induced Chemotaxis

1. In Vitro Macrophage Attraction Assay

1. Preparation of Conditioned Media

1. Warm up the tumor cell maintenance medium at a 37 °C water bath for 20 min.
2. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Take the cell detachment solution from the fridge. Spray the bottles of the medium and the cell detachment solution with 70% ethanol, wipe off 70% ethanol on the surface with paper towels, and bring them into the tissue culture hood.
3. Take the 6-well plates out from the incubator. Carefully pipette the conditioned media into 15 mL sterile centrifuge tubes. Put the tubes on ice. Pipette the media in the "media only" well into a separate 15 mL sterile centrifuge tube.
   NOTE: Before this step, make sure to check under the microscope to see if the cells attach. If not, one can use coated plates to allow better attachment or include the floating cells in the counting step.
4. Add 0.5 mL of cell detachment solution into each well of the 6-well plate. Check if the tumor cells round up every minute. Once the tumor cells round up, gently tap the side of the plate to help the cells detach.
5. Pipette 2.5 mL of tumor cell maintenance medium into the well, pipette up and down 3 times to mix and transfer the cells into a 15 mL sterile centrifuge tube. Take 10 μL of the cells and mix with 10 μL of Trypan Blue solution. Pipette 10 μL of the mixture into the counting slide and quantify the number of living cells using an automatic cell counter.
6. Adjust the volume of the conditioned media according to cell number using the serum free stem cell medium (37 °C incubation overnight). For example, if Well A has 3x as many live cells as the well with the least number of cells, dilute its conditioned media 1:3. This step is used to control difference caused by cell proliferation rate.
   NOTE: The reason to use serum free stem cell medium with 37 °C incubation overnight is to control the possible change in the media with prolonged 37 °C exposure.
7. Filter the conditioned media through 0.45 μm filters. Put the conditioned media on ice.
   NOTE: This step removes cells and cell debris in the conditioned media.

2. Preparation of MV-4-11 Cells

1. Warm up the IMDM medium (without FBS) at a 37 °C water bath for 20 min.
2. Spray the tissue culture hood surface with 70% ethanol and wipe off 70% ethanol on the surface with paper towels. Spray the medium bottle with 70% ethanol, wipe off 70% ethanol on the surface with paper towels and bring them into the tissue culture hood.
3. Take the flask of MV-4-11 cells into the tissue culture hood. Pipette 10 mL of cells into a 15 mL sterile centrifuge tube. Take 10 μL of cells and mix with 10 μL of Trypan Blue solution. Pipette 10 μL of the mixture into the counting slide and quantify the number of living cells using an automatic cell counter. Centrifuge the cells at 200 x g for 5min at 4 °C. Aspirate the supernatant.
4. Wash the cells with 10 mL of PBS and centrifuge the cells at 200 x g for 5 min at 4 °C. Aspirate the supernatant.
5. Resuspend the cells in IMDM medium (without FBS) for a final cell density of 1x10⁶ cells/mL.
   NOTE: The cell number of macrophages used here can be adjusted.

3. Transwell Assay

NOTE: For this step, use a cell migration assay kit or purchase the reagents and insert separately. For MV-4-11 cells, choose Transwell inserts with 5 μm pore size. To detect macrophage migration, directly quantify the number of macrophages in the lower chamber or use colorimetric/fluorimetric methods. Here, we demonstrate the assay using the colorimetric method.

1. Bring the 24-well plate, the insert and the reagents to room temperature.
2. Add 250 μL of MV-4-11 cells prepared above into each insert.
3. Add 400 μL of conditioned medium/medium only (with overnight incubation at 37 °C, these samples serve as the negative control) to the lower chambers of the 24-well plate. For each tumor line or control, use triplicate samples. Avoid bubbles between the insert and the conditioned medium. Bubbles will inhibit macrophages from migrating to the bottom wells.
4. Incubate the plates in a 37 °C incubator with 5% CO₂ for 4 h.
   NOTE: The incubation time can be adjusted. Cells migrating into the lower chambers can be seen under the microscope.
5. Gently tap the insert on the inner wall of the same well and discard the insert. As MV-4-11 cells grow in suspension, cell detachment solution or trypsin treatment is not needed here.
6. Gently pipette the cells in the wells up and down 3 times to mix. Transfer 225 μL of cell suspension to a black-walled 96-well plate suitable for fluorescent measurement.
7. Dilute the CyQuant dye 1:75 with 4x lysis buffer. Vortex briefly and spin down the solution. Add 75 μL of solution to each well of the 96-well plate. Incubate 15 min at room temperature.
8. Read fluorescence using the 480/520 nm filter set with a fluorescence plate reader.
9. Analyze data by subtracting the blank and normalizing to the control tumor cell line.