All antibodies were incubated using the manufacturers’ recommendation of 1 hour at room temp. Primary CTC panel: FITC labeled-anti-Cytokeratin 8, 18, 19; r-Phycoerythrin (PE) labeled anti-EpCAM; and Cyanine5 labeled anti-CD45 (Creatv MicroTech)1 (link)2 (link)3 (link); second panel: Alexafluor 488 labeled anti-PDL1 (2.5 ug/mL) and Dylight 650 labeled PD-1 (5 ug/mL) both PD-L1 and PD-1 were gifts from Dr. Steven Lin, MD Anderson Cancer Center, PE labeled CD34 (2.5 ug/mL, clone 4H11) and third panel: FITC labeled anti-CD14 (5 ug/mL, clone 61D3), PE labeled anti-CD184 (5 ug/mL, clone 2B11), efluor660 labeled anti-Vimentin (2.5 ug/mL, clone V9).
An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to image the samples. Exposures were preset as 2 sec (Cyanine5 and APC), 2 sec (PE), 1000 msec (FITC and Alexafluor 488), 500 ms (efluor660), and 10–50 msec (DAPI) for equal signal comparisons between cells. A Zen2011 Blue (Carl Zeiss) was used to process the images, mark the x/y placement of the cells and relocate previously imaged cells.
An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to image the samples. Exposures were preset as 2 sec (Cyanine5 and APC), 2 sec (PE), 1000 msec (FITC and Alexafluor 488), 500 ms (efluor660), and 10–50 msec (DAPI) for equal signal comparisons between cells. A Zen2011 Blue (Carl Zeiss) was used to process the images, mark the x/y placement of the cells and relocate previously imaged cells.
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