Immunofluorescence Assay: A Method to Identify Tumor Cells Captured on a Medical Wire

Abstract

Source: Gallerani, G. et.al., Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH. J. Vis. Exp. (2017).

This video describes the technique of immunofluorescence staining of circulating tumor cells captured on a functionalized medical wire. The functionalized wire allows isolation of CTCs in vivo, directly from patient’s blood stream. 

Protocol

1. Immuno-DNA FISH on Wire

1. Cell line spike-in on wire (3D support)
   NOTE: Preliminary set-up involves an accurate selection of adherent cells lines based on EpCAM-positivity. The wire is functionalized with anti-EpCAM antibodies so that only EpCAM-positive cells are stained.
   NOTE: Do not touch the functionalized part of the wire to avoid any damage.
   NOTE: All steps should be carried out under a laminar flow hood in sterile conditions.
   1. Resuspend the entire contents of a T75 flask (80% confluence) in a 5 mL vial using 4 mL of complete medium; for a single spike-in, about 2,000,000 cells are recommended to maximize cells adhesion to the wire.
   2. Remove the wire from its glass packaging.
   3. Dip the functionalized gold part of the wire into the vial, ensuring that the wire-stopper is a watertight fit for the vial. Seal with laboratory film.
      NOTE: The use of 5 mL tube is recommended to allow for a correct match between the wire-stopper and the vial.
   4. Incubate for 30 min at RT in a tube rotator (0-120 angle).
   5. Rinse the wire three times in 1× PBS solution using 3 different clean vials.
2. Fixation and permeabilization
   CAUTION: Acetone is a flammable substance that can irritate the respiratory tract. Only use acetone-resistant plastics or glass containers (no PVC or PVDF).
   NOTE: Perform these steps under a chemical fume hood.
   1. Air-dry the wire.
   2. Fix the cells by dipping the wire in acetone 100% solution for 10 min at RT. Use a 5 mL tube.
      NOTE: The wire-stopper must not come into contact with the fixative.
   3. Air-dry the wire for 5 min at RT.
      NOTE: The protocol can be paused here. The wire must be stored at -20 °C. When packing the wire for long-term storage, place the wire-stopper next to the functionalized tip and carefully insert the wire into the storage glass tube, taking care not to damage the functionalized tip. Close the storage glass and store (vertically or horizontally) at -20 °C.
3. Immunofluorescence Day 1
   NOTE: This stage involves an overnight incubation (~ 16 h).
   1. Wash twice in 1× PBS solution using 5 mL tube.
   2. Incubate the wire in antibody dilution buffer for 30 min at RT using 5 mL tube.
   3. Prepare 150 µL of antibody mix with a dilution of monoclonal primary antibody conjugated with fluorochrome in antibody dilution buffer, as specified in the datasheet. For example, EpCAM-FITC antibody was used with a 1:20 dilution.
   4. Use a p200 tip to perform incubation, as follows: 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.
   5. Gently drop 150 µL of the antibody mix (for example anti EpCAM_FITC antibody 1:20 diluted) into the tip. To avoid the risk of bubble formation, gently twirl the wire until the functionalized part is completely plunged.
   6. Seal up the hole of the tip. Wrapping laboratory film around the hole can help.
   7. Incubate vertically overnight (~ 16 h) in the dark at 4 °C.
4. Immunofluorescence day 2
   1. Block antibody incubation by washing the wire twice in 1× PBS.
   2. Re-insert the wire in its wire-stopper.
      NOTE: Store in 1X PBS at 4 °C in a 5 mL vial until the FISH assay is performed.

Materials

Ethanol	Carlo Erba	# 414605
Tween 20	BIO RAD	# 1706531
PBS (Phosphate Buffered Saline)	Medicago	# 09-9400-100
Acetone	Sigma Aldrich	# 534064-500ML
RUBBERCEMENT	Royal Talens	# 95306500
EpCAM-FITC (clone: HEA-125)	Mitenyi	#130-080-301
RPMI 1640	Gibco	# 31870-025
XT ALK BA	MetaSystems Probes	# D-6001-100-OG
Detektor CANCER01 EpCAM	Gilupi Nanomedizin		Functionalized wire
ZEISS Fluorescent Microscope Axioskop	ZEISS
Nikon NIS-Elements BR 4.11.00 64 bit software	Nikon
Nikon DS-QiMc 12 bit digital camera	Nikon	DS-QiMc