CTC Immunofluorescence Assay : A Technique to Characterize Circulating Tumor Cells
Published: April 30, 2023
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 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…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
Citar este artículo
CTC Immunofluorescence Assay : A Technique to Characterize Circulating Tumor Cells. J. Vis. Exp. (Pending Publication), e20231, doi: (2023).