Materials and Methods
Whole blood was obtained from healthy volunteers and placed in tubes containing EDTA. Red blood cells (RBC) were then lysed using RBC lysis buffer and the pellet was suspended in wash buffer (phosphate-buffered saline [PBS] with 2% fetal bovine serum [FBS]). SKBR-3 cells were cultured in flasks with Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS before subsequent harvesting using trypsin.
Cell count was determined for WBCs and SKBR-3 cells using a Scepter™ handheld cell counter (EMD Millipore). The WBCs were plated in a 24-well plate and SKBR-3 cells were spiked into the WBC sample such that the final ratio of SKBR-3 cells to WBCs was 1:100,000.
EPCAM Cy-5 and Her-2 Cy-3 SmartFlare probes were then added to individual wells at a concentration of 25 pM. The probes enable target RNA levels to be determined in live, individual cells while leaving those cells intact for further analysis. The technology uses gold nanoparticles bound to sequence-specific oligonucleotide probes; in the presence of their targets, the probes fluoresce, allowing the cells to be imaged and the RNA quantified using flow cytometry. The probes enter and exit the cell through natural endocytosis and exocytosis without adverse effects, making them a valuable tool for studying CTCs within peripheral blood mononuclear cells (PBMCs).
SmartFlare uptake control, which has a constitutively fluorescent fluorophore, was used as the positive control probe. SmartFlare scramble control, which targets nonsense mRNA sequences not present in cells, was used as the negative control. Cells were incubated with the probes for 16 hours.
After incubation, cells were harvested, centrifuged, and resuspended in 50 uL of wash buffer. Analysis was performed on the ImageStreamX Mark II flow cytometer, which has the capacity to acquire multispectral images of large numbers of cells. Image analysis was performed using the image-based algorithms available in IDEAS 6.0 image analysis software.