Non-permeabilized (A) and permeabilized cells (B) were stained with anti-HA FITC-conjugated antibody as described above. immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration Melphalan of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of CD140a ion channels that can be extended to study ion pumps and plasma membrane transporters. laser and optics are performing to specification, the laser and flow cell are properly aligned) by using instrument setup beads. Use the 100 m nozzle with 20 psi sheath pressure. Melphalan NOTE: The nozzle does not have to be changed on a bench flow cytometer. Set the cytometer’s flow rate according to the manufacturer specification. Exceedingly high flow rates will decrease sensitivity in the detection of variations in fluorescence. Select blue (488 nm to excite Fluorescein Isothiocayanate or FITC) and yellow-green (561 nm to excite mCherry) lasers. Collect FITC and mCherry fluorescence levels with a 530/30 nm and with a 610/20 nm bandpass filter respectively. Acquire the forward scatter (FCS) versus side scatter (SSC) dot plot for unstained cells using linear scale. Adjust each detector’s amplification to visualize cells in the lower left quadrant of the dot plot. Sample Reading of Intact Non-permeabilized Cells Set the P1gate for live non-permeabilized cells by delineating a free form around the cells to be analyzed excluding cell debris and cell aggregates, thus limiting the fluorescence signal to intact cells. NOTE: Live/dead exclusion dyes can be used to facilitate gate placement on live cells. Set 10,000 events to record in the stopping gate P1. Set this to a higher number of events if need be. Acquire mCherry versus FITC two-parameter contour plot to detect baseline autofluorescence of unstained cells. Use bi-logarithmic scale to show negative values and improve resolution between populations25. Adjust each detector’s voltage to set the Melphalan unstained negative cells within the lower portion of the first ten units of the log fluorescence intensity plots. Acquire all intact non-permeabilized samples using settings established in 4.1.5 and 4.1.6 and collect FSC, SSC and signals in the fluorescence detectors. Export and save *.fcs files to be used for analysis using flow cytometry analysis software. Sample Reading of Permeabilized Cells Move the P1 gate to select live cells in the permeabilized samples and adjust FSC and SSC voltage as shown in 4.1.5 and 4.1.6. Acquire all permeabilized samples and collect FSC, SSC and signals in the fluorescence detectors. Export and save *.fcs files to be used for analysis using flow cytometry analysis software. Data Analysis Melphalan Launch the flow cytometry analysis software and import *.fcs files saved in 4.2.4 and 4.3.3. Click on the first sample listed in the workspace window. A new window named after the tube I.D. number opens automatically. Start the gating process in the plot of SSC versus FSC. Draw a gate (P1) using the Ellipse icon around live cells and eliminate any debris, dead cells, or aggregates which have different forward scatter and side scatter than live cells To draw the two-parameter contour plot of the mCherry (y-axis) versus FITC (x-axis) fluorescence intensity of the live cells, click first on the x-axis and choose the FITC-A channel and then click on the y-axis and choose the PE-mCherry-A channel. Click on the “Quad” icon to position the quadrant marker.