Metamorph image analysis system

For the evaluation of Pgp expression, MP pellets were prepared from 50 cancer patients. Briefly, plasma containing MPs was centrifuged at 4,200 × g at room temperature for 10 min and the MP pellet was cytocentrifuged at 400 × g at room temperature for 10 min (Shandon CytoSpin 2 Cytocentrifuge; Thermo Fisher Scientific, Inc., Waltham, MA, USA). To assess MP samples for CD243 expression, the Histostain^®-Plus 112 kit was used (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Briefly, slides were fixed for 10 min at room temperature with 3% paraformaldehyde in Dulbecco's 1X PBS (pH 7.6). To prevent non-specific binding, slides were incubated for 10 min with serum blocking solution, followed by incubation for 45 min at room temperature with murine anti-CD243 monoclonal antibody (1:20). Following washing three times with 1X PBS, slides were incubated for 10 min with the enzyme conjugate provided in the kit. Following further washing with PBS, samples were incubated for 10 min with 3,3′-diaminobenzidine, washed with distilled water and coverslipped with mounting medium. Subsequently, the slides were observed under a Zeiss Axioscope light microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a CoolSNAP camera and the MetaMorph Image Analysis system (Universal Imaging Corp, Downingtown, PA, USA).