- To begin, dilute the isolated circulating tumor cells or CTCs to the required concentration using an anti-binding solution that protects cells from fluid shear and pressure damage. Next, assemble a poly-lysine coated microscope slide and a cytocentrifuge filter card into a cytocentrifuge funnel assembly. Transfer the cell suspension into the funnel port of the assembly followed by cytocentrifugation.
The centrifugal force concentrates and deposits a monolayer of cells onto a circular area on the slide. Isolate the area deposited with cells using a silicone isolator. Once dry, fix the cells using paraformaldehyde. Next, incubate the cells with a suitable blocking solution containing proteins that bind readily to any non-specific binding sites on the cell surface.
Now, label the cells with a fluorescent-labeled antibody solution of interest. The fluorophore-tagged antibodies recognize and bind to specific target molecules on the cell surface. Wash the sample to remove any unbound antibodies. Add a mounting solution to prepare the sample for imaging.
Use a fluorescence microscope to detect the light emitted by the antibody-fluorophore conjugates bound to the target antigens on CTCs. In this protocol, we will perform immunofluorescence assay of CTCs in non-small cell lung cancer patient samples.
- For immunofluorescence staining, count the cells in a hemocytometer, and dilute the enriched sample to a 1 times 10 to the fifth cells per 100 microliters of 0.2% anti-binding solution concentration. Next, use a cotton swab soaked with 50 microliters of anti-binding solution to moisten the contour of the sample chamber and place a poly-lysine glass slide in the sample chamber.
Close the chamber, and pipette up and down three times to coat a micropipette tip with the binding solution before re-suspending the sample in the last 100 microliters of the supernatant. Transfer the cell solution to the sample chamber and cytospin the sample in a dedicated centrifuge at 400 rotations per minute for 4 minutes at a low acceleration.
At the end of the centrifugation, place a silicon isolator around the area of deposition and let the slide dry under a microbiological safety cabinet for two minutes. Then, fix the cytospun sample with 100 microliters of 4% paraformaldehyde for 10 minutes at room temperature, followed by three 2 minute washes with 200 microliters of PBS per wash at room temperature.
After the last wash, block any non-specific binding with a 30-minute incubation in blocking reagent at room temperature followed by labeling with 100 microliters of the antibody solution of interest. Place the labeled slide in a 100 by 15-milliliter Petri dish on a piece of absorbent paper moistened with 2 milliliters of sterile water and placed the dish closed at 4 degrees Celsius overnight, protected from light.
The next morning wash the sample three times with PBS as demonstrated and mount the sample with 10 microliters of an appropriate mounting solution and a glass cover slip. Then seal the cover slip with clear nail polish.