The overall goal of this method is to quantify the cell surface expression of membrane proteins using a fluorescent spaced assay. The main advantage of this technique is that flow cytometry assays deliver quantifiable end points on large volumes of live cells in a single experiment. The implications of this technique extend toward a diagnosis and a therapy of cardiac ventricular arrhythmias because these pathologies are often associated with genetic mutations that cause defects in the trafficking of cardiac ion channels.
Generally individuals new to this method struggle to identify a suitable extrorsal insertion site for the epitope that does not hurt their protein function and generate a strong fluorescent signal in the presence of the conjugated antibody. Demonstrating this procedure will be Benoite Bourdin, a research assistant from my lab. To begin, doubley tag DNA constructs with mCherry at the intracellular C-terminus.
Also, express an extra cellular facing hemmagglutinin epitope, which can be measured using a fluorescence antibody to estimate the cell surface expression of membrane proteins. Next, culture a plate of TSA201 cells using standard techniques until they reach 90%confluence. For each sample prepare the transfection reagents in two tubes.
Prepare one 1.5 milliliter tube with four micrograms of DNA and 250 microliters of reduced serum medium, and a second tube with 10 microliters of the liposome-mediated transfection reagent and 250 microliters of serum reduced culture medium. Gently mix the two tubes and incubate them for five minutes at room temperature. Then, gently combine the contents of tube one and tube two and incubate the liposome DNA complexes for 20 minutes at room temperature.
Add the complexes to the cultured cells and gently rock the culture dish. Place the dish back at 37 degrees Celsius and under a 5%carbon dioxide atmosphere for 24 hours. 24 hours after the addition of the transfection reagent, remove the medium from the culture dish and carefully wash the cells with 400 microliters of pre-warmed Trypsin-EDTA.
Then, add 400 microliters of Trypsin-EDTA and incubate the dish for five minutes to allow cells to detach from the dish. Once detached, stop the enzyme digestion by adding one milliliter of cold culture medium without penicillin or streptomycin to the cells. Gently pipette the medium over the plate four to five times to wash all the cells from the surface.
Then, collect the cells in sterile 1.5 milliliter tubes and immediately place them on ice. For the next steps, use ice-cold solution and keep the cells at four degrees to prevent the internalization of the surface epitope. Also, decrease the cells exposure to light in order to limit the photobleaching of the fluorescent signal.
Next, centrifuge the tubes to pellet the cells. Then, carefully aspirate and discard the resulting supernatant. Resuspend the pellet to prepare a single cell suspension using one milliliter of PBS.
Then, briefly centrifuge the tubes. Pellet and resuspend the cells once more in order to completely remove the culture medium. Next, resuspend the cell pellet in 600 microliters of PBS.
Measure the cell concentration and adjust it to at least three million cells per milliliter. Then, divide the cells into new 1.5 milliliter tubes. Be sure to include the appropriate controls to discriminate specific staining from non-specific staining and for extra cellular staining and also for intracellular staining.
The isotope control antibody will help to assess the level of background staining and should match the primary antibody, auspicious isotope and fluorophore. It is important to use the control isotope antibody and the fluorescent antibody at the same concentration. To estimate the cell surface expression of membrane proteins, first label just the extra cellular facing HA epitope using a fluorescent light conjugated antibody.
Then, add a FITC-conjugated monoclonal anti-HA antibody at five micrograms per milliliter to the tube. Vortex the cells briefly and then incubate them on a rocker platform in the dark. Remove the cells from the dark and add 900 microliters of PBS.
Then centrifuge the tube to pellet the cells and aspirate the supernatant. Next, wash the cells by resuspending the pellet in one milliliter of PBS and briefly vortexing the cells. Then, repeat the centrifuge step to again pellet the cells.
Repeat this wash step a total of three times to remove any unbound antibody. After the final wash, resuspend the cells in 500 microliters of PBS and transfer the single cell suspension to a five milliliter flow cytometry tube. Keep the cells in the dark at four degrees Celsius until the sample is run.
To estimate the intracellular expression of total protein next label the permeabilized cells. This is accomplished using a similar protocol to the extracellular labeling, but with the addition of a saponin based cell permeabilization buffer. Pellet the cells, discard the supernatant and resuspend the cells in 100 microliters of fixation permeabilization solution directly from the prepared stock.
Incubate the cells in the dark at four degrees Celsius for 20 minutes. Then, add 100 microliters of freshly prepared permeabilization washing buffer and briefly vortex the cells. Next, pellet the cells and wash them two more times using the permeabilization washing buffer.
Then, add the FITC-conjugated monoclonal anti HA antibody at 5 micrograms per milliliter and 100 microliters of the permeabilization washing buffer. Briefly the vortex the cells and incubate the cells in the dark at four degrees Celsius for 30 minutes. Next, add 100 microliters of the permeabilization washing buffer.
Pellet the cells and wash them three times in 100 microliters of the same buffer as previously shown. After the final wash, resuspend the cells in 500 microliters of PBS and transfer the single cell suspension into a five milliliter flow cytometry tube. Run the non-permeabilized and permeabilized cells through the flow cytometer on the same day.
To begin, launch the flow cytometry analysis software and import the fcs files created during flow cytometry. Click on the first sample and start the gating process in the plot of side scatter versus forward scatter. Next, draw the P1 gate using the ellipse icon around the live cells to eliminate any debris, dead cells or aggregates.
Then, draw the tube parameter contour plot of the mCherry versus the FITC fluorescence intensity. Set the P2 gate around the FITC and mCherry positive cells and the P3 gate around the fluorescence negative cell population. Next, select the P2 and P3 gates and click on the add statistics icon in the original workspace window.
Click on count and then click on mean. Finally, apply the gates parameters and statistics to all samples probed by the cytometer. This protocol was used to characterize the role of N-glycosylation on total and cell surface expression of CaV Alpha 2 Delta 1 in TSA201 cells.
To access the cell surface protein expression HA was measured on non-permeabilized cells that had N-glycosylation sites disrupted at four sites, 16 sites or no site. The FITC levels were strong in the wild type and cells with N-glycosylation cells disrupted at four locations, but only background levels were found in the cells with 16 locations disrupted. For the permeabilized cells, the FITC fluorescence increased for all conditions, indicating that all transfected, HA-tagged, CaV Alpha 2 Delta 1 proteins were stained and detected by the anti HA FITC antibody.
In addition, the mCherry fluorescence levels were similar for permeabilized and non-permeabilized cells, signifying that the permeabilization does not alter the relative mCherry signal. Assays conducted after cell permeabilization showed that total protein expression for the cells with 16 locations disrupted was also significantly decreased, whether it was inferred from the FITC fluorescence in permeabilized cells or from the constitutive mCherry fluorescence measured in non-permeabilized and in permeabilized cells. In addition to this procedure, extra physiological recordings can be performed in order to correlate expression and function of ion channels.
This technique also paves the way to researchers in the medical field to study the impact of genetic mutations, posttranslational modifications, microRNAs, small GTPases and the role of chaperones in the trafficking of membrane proteins expressed in recombinant or in native cells. After watching this video you should get a good understanding on how to perform DNA transfection in recombinant cells, to stain NTAC and permeabilized cells, as well as to perform data analysis on the samples. Don't forget that working with live cells requires precautions such as working in the helaminal fluid for the transfixion and wearing gloves when handling chemicals.
