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.
1. Immunofluorescence Staining
- 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.
- 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.
- 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 chamber. Centrifuge with a dedicated centrifuge (Table of Materials) at 400 rpm for 4 min (acceleration low).
- Place a silicon isolator around the area of deposition. Let dry the glass-slide under a microbiological safety cabinet for 2 min.
- Prepare the fixation solution by diluting 1 mL of 16% paraformaldehyde (PFA) with 3 mL of sterile PBS. Add 100 µL of fixation solution (4% PFA) per sample and incubate at RT for 10 min. Remove fixation solution and perform three washes with 200 µL of PBS and incubate at RT each for 2 min.
CAUTION: Use PFA under a chemical safety cabinet to prevent inhalation.
- Prepare the saturation solution by diluting the fetal bovine serum (FBS) at 5%, Fc receptor (FcR) blocking reagent at 5%, and bovine serum albumin (BSA) at 1% in sterile PBS (Table of Materials). Add 100 µL of saturation solution per sample and incubate for 30 min at RT. Remove saturation solution.
- Add 100 µL of antibody solution per sample (CD45 antibody 1/20; PanCK antibody 1/500; PD-L1 antibody 1/200; Qsp saturation solution 100 µL) (Table of Materials). Place the polylysine glass-slide in a 100 mm x 15 mm Petri dish. Moisten an absorbent paper with 2 mL of sterile water and close the Petri dish with the lid. Place at 4 °C overnight and protect from the light.
- Remove the antibody mix and perform 3 washes with 200 µL of PBS incubating each wash for 2 min. Let the sample dry for 5 min and protect it from the light. Place 10 µL of mounting solution (Table of Materials) in the area of deposition and cover with a microscope coverslip without making a bubble. Seal the coverslip with nail polish.
|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|