CTC Immunofluorescence Assay : A Technique to Characterize Circulating Tumor Cells

Abstract

Source: Garcia, J., et al. Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence. J. Vis. Exp. (2019)

This video describes the Circulating Tumor Cells (CTC) Immunofluorescence assay, a technique to characterize the biomarkers on circulating tumor cells in non-small cell lung cancer patient samples. This assay could be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and possible immunotherapy treatments.

Protocol

1. Immunofluorescence Staining

1. Enumerate on a chambered slide with a hemocytometer-type grid the number of cells per mL. Dilute the enriched sample with 0.2% anti-binding solution (Table of Materials) to a concentration reaching 100,000 cells/100 µL per cytospin.
2. Moisten the contour of the sample chamber using cotton (Table of Materials) with 50 µL of 0.2% anti-binding solution. Place a polylysine glass-slide in the sample chamber and close.
3. Coat a tip with 0.2% anti-binding solution by pipetting up and down 3x. Resuspend the enriched sample and transfer the cell solution into the sample chamb....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pluronic F-68 10%	Gibco	24040-032	Anti-binding solution. Storage conditions: Room temperature
EZ Cytofunnels	ThermoFisher	A78710003	Sample chamber with cotton. Storage conditions: Room temperature
Shandon Cytopsin4 centrifuge	ThermoFisher	A78300003	Dedicated centrifuge. Storage conditions: Room temperature
Paraformaldehyde 16%	ThermoFisher	11490570	Fixation solution. Storage conditions: +4°C
Phosphate Buffered Saline Ultra Pure Grade 1X – 1L	1st Base Laboratory	BUF-2040-1X1L	Storage conditions: Room temperature
FcR blocking Agent	Miltenyi Biotec	130-059-901	Storage conditions: +4°C
PD-L1 monoclonal antibody (clone 29E2A3) - Phycoerythrin	BioLegend	BLE329706	Storage conditions: +4°C
PanCK monoclonal antibody (clone AE1/AE3) Alexa Fluor 488	ThermoFisher	53-9003-80	Storage conditions: +4°C
CD45 monoclonal antibody (clone HI30) Alexa Fluor 647	BioLegend	BLE304020	Storage conditions: +4°C
Fluoromount	Sigma	F4680	Mounting solution. Storage conditions: Room temperature
Petri Dish	Dutscher	632180	Storage conditions: Room temperature
Phosphate Buffered Saline (PBS)	Ozyme	BE17-512F	Storage conditions: +4°C
Polylysine slides	ThermoFisher	J2800AMNZ	Storage conditions: Room temperature
Silicon Isolator	Grace bio-Labs	664270	Storage conditions: Room temperature
Sterile Deionized Water – 100 mL	1st Base Laboratory	CUS-4100-100ml	Storage conditions: Room temperature
Fetal Calf Serum (FCS)	Gibco	10270-106	Storage conditions: +4°C