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

In response to certain chemical attractants released by solid tumors, immune cells such as tumor-associated macrophages or TAMs infiltrate the solid tumors. Subsequently, these TAMs facilitate tumor progression.

To study the interactions between TAMs and tumor cells in vitro, begin by taking a multiwell plate with a membranous insert. This arrangement temporarily separates the well into two compartments.

Next, add a suspension of TAMs to the upper compartment of the transwell.

Supplement the lower compartment of the transwell with a conditioned medium enriched with chemical attractants secreted by tumor cells.

Incubate the plate for the desired time.

During incubation, chemical modulators attract TAMs. This leads to the movement of TAMs into the lower compartment via the porous insert membrane through chemical-induced chemotaxis.

Now, remove the insert from the well. Transfer the conditioned medium containing the migrant TAM population into a suitable plate for fluorescent measurement.

Treat the cells with a lysis buffer containing fluorescent dye. This buffer lyses the macrophages. Eventually, the dye molecules bind to the nucleic acids, producing an enhanced fluorescence. 

Finally, scan the plate using a fluorescence plate reader.  A high fluorescence indicates the successful migration of macrophages through chemical-induced chemotaxis.

First, bring the necessary materials to room temperature. Add 250 microliters of the prepared MV-4-11 cells to each insert. Next, add 400 microliters of either the conditioned medium/medium only to the lower chambers of the 24-well plate, making sure to add the samples in triplicate.

Incubate at 37 degrees Celsius with 5% carbon dioxide for 4 hours. Then, gently tap the insert on the inner wall of the same well and discard the insert. Gently pipette the cells in the wells up and down three times to mix.

Transfer 225 microliters of this cell suspension into the wells of a black-walled 96-well plate suitable for fluorescent measurement. After this, dilute the CyQuant dye with 4x lysis buffer at a ratio of 1:75. Vortex briefly and spin down the solution.

Transfer 75 microliters of this solution to each well of the 96-well plate and incubate at room temperature for 15 minutes. Using a fluorescence plate reader, read the fluorescence and analyze the data as outlined in the text protocol.