Multi-Color Flow Cytometric Analysis Using UV-Excited Fluorochromes

Study on the Expression of CD98 amino-acid transporter.

Abstract

To apply UV-excited fluorochromes to a BD LSR benchtop flow cytomerty system, we examined fixation and staining conditions using UV-excitable dyes such as Hoechst 33342, DAPI and Indo-1. The BD LSR is available as a two-laser system featuring an argon laser (488 nm) and an UV-laser (325 nm). Flow cytometric analysis of DNA content with DAPI without RNase-treatment exhibited lower CV value of G₀/G₁ phase peak than that with PI. Analysis of human tumor cells and mouse lymphocytes for simultaneous staining of DNA and cell surface markers such as CD98 revealed that fixation with 2-4% paraformaldhyde followed by permeabilization with 0.1% saponin give the best result. Flow cytometric analysis of DNA content in intact cells treated with Hoechst 33342 displayed a CV value of G₀/G₁ phase peak comparable to fixed cells. Thus, we established simultaneous analysis of DNA content and multiple surface markers by using UV-excited fluorochromes and BD LSR cytometer. Next, we used BD LSR to measure intracellular Ca²⁺ concentration in murine thymocytes that had been loaded with the Ca²⁺-sensitive, UV-exitable dye Indo-1. We could demonstrate the elevation of intracellular Ca²⁺ after CD3-stimulation characteristically in CD4⁺CD8⁺ and CD4^-CD8^- subsets. Thus,the combined application of UV-excited dye, Indo-1 and BD LSR enable us to simultaneously analyze intracellular Ca²⁺ and various cell surface markers.