Epics xl mcl flow cytometer

Tissue samples wetted with 1 ml 1X PBS were mechanically disaggregated by placing them into a Medicon (BD Biosciences) and inserted in the Medimachine (BD Biosciences) for 45 sec at 100 rpm. Cell suspensions were filtered using a Filcon (BD Biosciences), washed twice with 1X PBS and divided at a concentration of approximately 1×10⁷ cells/ml into aliquots for staining. For detection of mCD44v6 positive cells, cell aliquots were incubated with CD44v6 (Bio-Rad MCA1730, dilution 1:500) for 1 h at room temperature (RT) in an orbital shaker at low speed. Cells were then washed and the percentage of cells expressing mCD44v6 was detected following a 60 min incubation with a goat anti-mouse IgG1: FITC secondary antibody (Bio-Rad STAR132F; dilution 1:100).
For intracellular labelling, cells fixation-permeabilization was performed using 1% paraformaldeyde-1% saponin in 1X PBS for 20 min at 4°C before cells were incubated with MTA1 antibody (SantaCruz Biotechnology SC-17773 dilution 1:500) and stained with a Polyclonal Goat anti-mouse-RPE Goat F(ab)2 secondary antibody (Dako R0480, dilution 1:100). Unstained cells were processed in parallel and included as references during analysis as well as cells stained only with the secondary antibodies. Flow cytometry analysis was performed using a Coulter Epics XL-MCL Flow Cytometer and its integrated SYSTEM II™ Software.

To examine morphological changes of erythrocytes by pressure, reserved erythrocytes at a 10% hematocrit in PBS were incubated for 0 and 24 h at 37°C and exposed to a pressure of 200 MPa for 30 min at 37°C [17 (link)]. After the decompression up to 0.1 MPa (atmospheric pressure), the suspensions were used as samples of a light microscope (model IX-71, Olympus).
Pressure-induced hemolysis was performed as previously described [17 (link),22 (link)]. Briefly, the erythrocytes (or red ghosts) suspended at a 0.3% (or 0.6% for red ghosts) hematocrit in PBS were exposed to a pressure of 200 MPa for 30 min at 37°C. After the decompression, the erythrocyte suspensions were centrifuged for 1 min at 3,000×g and room temperature. The absorbance of the supernatants was measured at 542 nm. The particles produced from erythrocytes by high pressure were analyzed using an EPICS XL-MCL flow cytometer (Coulter, USA) [21 (link),22 (link)].

Single- and dual-color immunophenotyping of lymphocytes was performed at 48hr as described in our previous studies16 (link),29 (link) with modifications for intracellular staining. Gating strategies included forward and side scatter, single parameter histograms, two-parameter density plots, and back-gating to confirm gating strategies. Antibodies (Abs) used included mouse anti-human monoclonal antibodies (mAbs) (of the IgG1 isotype): fluorescein isothiocyanate (FITC)-conjugated CD3, CD14, CD45RO, CD45RA, CD154, CCR7, IL-2, IL-4, IFN-gamma; phycoerythrin (PE)-conjugated CD8 and CD4-perCP; and the following Simultest (FITC/PE-conjugated) reagents: CD4/CD8, CD3/CD19. All mAbs were purchased from BD Biosciences (San Diego, CA) or R&D Systems (Minneapolis, MN), and titrated to obtain maximum staining efficiency according to manufacturer’s recommendation. Flow cytometric analysis was performed on a Coulter Epics XL/MCL Flow Cytometer using System II software (Coulter, Miami, FL) and CytoComp (Coulter). The total numbers of lymphocytes were calculated from the white blood cell (WBC) count (total lymphocytes/mm³ or percentage total lymphocytes).