Immunofluorescence Assay: A Method to Identify Tumor Cells Captured on a Medical Wire
Transcripción
– Take a functionalized wire having a small gold portion coated with a hydrogel coupled with antibodies of interest. Dip the gold part of the wire in a vial containing tumor cells. Close it with a wire stopper, and incubate it in a tube rotator. The circulating tumor cells or CTCs displaying surface antigens bind to the antibodies on the wire. Rinse the wire by dipping in vials containing phosphate buffered saline or PBS and air dry. Now dip the wire in an acetone solution to fix the cells by dehydrating them.
Next, gently insert the wire through a P200 tip with the functionalized part within it. Fill the tape with an antibody conjugated with a fluorochrome to submerge the wire. These labeled antibodies will bind to their respective antigens present on the cells. Seal the opening with a laboratory film and incubate overnight in dark at 4 degrees Celsius. Next, wash the wire with PBS to remove any unbound antibodies. Place the wire into a wire holder and view under a fluorescent microscope. The CTCs bound by antibodies fluoresce on the wire. In the following protocol, we will capture CTCs on a medical wire and identify them by immunofluorescent staining.
– To begin the experiment, re-suspend the entire contents of a T75 flask that is at 80% confluence in a 5 milliliter vial using 4 milliliters of complete medium. Next, remove the wire from its glass packaging. Dip the functionalized gold part of the wire into the vial ensuring that the wire stopper is a watertight fit for a 5 milliliter vial. Seal the wire with laboratory film. Then incubate the wire in a tube rotator for 30 minutes at room temperature. After the rotation, rinse the wire three times in one fold PBS solution using three different clean vials and wait for the wire to dry.
After air drying the wire for 10 minutes in a hood, fix the cells by dipping the wire in 100% acetone solution in a 5 milliliter tube for 10 minutes at room temperature. Ensure there is no trace of acetone on the functionalized tip. Air dry the wire for 5 minutes at room temperature. Next, in a 5 milliliter tube, wash the wire twice in one fold PBS. Incubate the wire in antibody dilution buffer.
Prepare 150 microliters of antibody mix with a dilution of monoclonal primary antibody conjugated with fluorochrome and antibody dilution buffer as specified in the data sheet. Then use a P200 tip to extract the wire from the wire stopper and gently insert the wire through the larger hole of the tip. Insert the unfunctionalized end first and slowly pull the wire through the smaller hole until the gold part is just beyond the larger hole. Gently add 150 microliters of the antibody mix into the tip.
To avoid the risk of bubble formation, gently twirl the wire until the functionalized end is completely plunged. Seal the hole of the tip and wrap laboratory film around the hole. Incubate the wire vertically overnight in the dark at 4 degrees Celsius. On the second day, block the antibody incubation by washing the wire twice in one fold PBS. Reinsert the wire in its wire stopper.
