Fluorescence Based Transwell Macrophage Attraction Assay: An In Vitro Method to Study Tumor-Macrophage Interaction 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.