With increasing interest in the role of cellular factors in HIV infection of macrophages, we've developed a flexible system of analysis, particularly for SAM HG one, which is not expressed in U937 cells. The main advantage of the system is its flexibility. Once it's set up, it can be used to analyze a variety of proteins, cells, or target viruses.
It's important to maintain the health of the cells and also to be comfortable with multicolor flow cytometry. Turn on the cytometer and computer 10 minutes before starting the analysis. Ensure that the waste is empty and the sheath tank is full.
Then log in and open the analysis software. After moving the arm, take the water tube off. Set the flow rate to high, and press prime.
Wait for the light to go out and repeat this process three more times. Place the water tube back and run at a high flow rate for three minutes. Then, set the flow rate to low and press standby until acquisition.
Select experiment, new experiment, and press okay to select blank experiment, right click on the experiment and add new specimen. Press okay to blank panel. Right click to rename with an appropriate name and open the specimen by clicking the plus sign to reveal a new tube.
Double click on the tube to see the cytometer settings in the inspector. In the parameters menu, delete the unrequired fluorochromes except the blue laser 53030 and yellow laser 61020. On the worksheet, create a forward scatter area versus side scatter area dot plot, YFP histogram, RFP histogram, and RFP versus YFP dot plot by clicking on the corresponding icon and changing the axes by clicking on the axis label.
Load the uninfected, untransduced control and press run on the machine. Then press acquire in the acquisition dashboard. Adjust the forward and side scatter voltages in the dashboard to position the cells in the lower left quadrant.
Gate around this population and label it as P one. Right click on the other plots and select show P one to remove the debris from the subsequent analysis. Adjust the RFP and YFP voltages down so that the peak sits on the left of the histogram.
Load the single color control for YFP and press acquire. Adjust the YFP voltage in the dashboard so that the most fluorescent cells are within the detector range. Then repeat the same for the RFP control.
Select the experiment. Go to the compensation setup and select create compensation controls and okay to toggle to a normal worksheet then select an unstained normal worksheet. Press on the run and acquire for the unstained control.
Draw a gate P one around the the intact cells and record. Remove the tube and put the machine on standby. Right click on P one.
Click on apply to all compensation controls and switch to the RFP normal worksheet. Press the run, and record the single color RFP control and the software will automatically gate the positive cells. Put the machine on standby and repeat the same for the YFP control.
Select experiment, go to compensation setup, select calculate compensation and then click on link and save. Switch to the global worksheet and return to the specimen tube. Acquire the cells transduced with wild type SAM HD one infected with HIV RFP, and check that four distinct populations can be seen in the corners of the quadrants aligned vertically and horizontally for YFP and RFP.
Remove the tube and press standby. Reload the untransduced, uninfected sample and press run. In the acquisition dashboard, set the stopping gate to P one and events to record 30, 000 events.
Press record. Repeat the same for other samples and controls by pressing next tube between each tube. Rename the samples while running or post-hawk, and export the data as dot FCS files.
Open the software and drag all FCS files into the dashboard. Select the compensation controls and drag them into the compensation sub folder. Double click on the untransduced, uninfected tube to open the file and forward scatter a versus side scatter a plot.
Select the polygon tool and gate on the intact cell population excluding debris in the lower left corner and name it cells. Move the label so it doesn't obscure the cells. Drag this gate to the whole population of tubes in the all samples bar.
Scroll through the whole population using the horizontal arrow buttons to ensure appropriate gating for each tube. Double click on the cells and adjust the axes to height versus area. Select the single large population using a rectangular gate to exclude doublets and to allow the software to suggest naming it as single cells automatically.
Then, drag this gate to all cells populations and check that the gating is appropriate for all samples. Select the single cells population for the uninfected, untransduced sample. Change the Y axis to compensated blue laser for YFP and X axis to compensated yellow laser for RFP.
Select the quadrant gating tool and click at the upper right extreme of the negative cell population. Apply this preliminary gating to all single cell populations by dragging onto the parent gate in the all samples bar. If the quadrant gates do not separate the population satisfactorily, gate the individual quadrants using the polygon tool.
Scroll to the wild type SAM HD one only sample to check the correct gating between the untransduced YFP negative and YFP positive cells. Use the contour view to discriminate the negative cells from dim YFP cells. Use the best division between YFP negative and positive that is valid for all samples and scroll through the samples to check the gating with respect to YFP positivity.
Scroll to the untransduced HIV RFP infected sample and check whether the gating between the uninfected RFP negative and infected RFP positive is correct. Use the contour view if required and scroll through the remaining samples to check that the gating is valid for all samples. Each sample requires four quadrants.
Q1 YFP positive, Q2 double positive, Q3 RFP positive and Q4 double negatives. Open the table editor and drag the four quadrant gates into the dashboard. Select to file and excel from the dropdown.
Choose your file destination and click on create table. Save the generated spreadsheet according to local file naming conventions. Click on the layout editor icon to export the representations of plots and gating strategies.
Select each population and drag it into the editor with the required axes, labeling and spacing. To create a layout with the same plots shown for all samples under the batch section enter one in the column and press create batch report. In the file menu, select the scale to width and avoid page breaks.
Then save in the required file format. In the exported spreadsheet, generate the columns as described in the text manuscript. Use appropriate data analysis software to average the replicate data for each SAM HD one construct, and calculate the restriction ratio values.
Representative YFP versus RFP plots were generated for optimal and suboptimal data. The suboptimal data plot indicates that the HIV infection was too low and created difficulties with compensation and gating. The restriction ratio was 0.5, which is higher than the expected ratio of 0.3.
The plots of restriction ratio for variants of SAM HD one with respect to the wild type and negative control were generated for the optimal and suboptimal data. In the optimal data, the wild type showed the expected restriction of approximately 0.2 and negative control HD 206 to seven AA had the restriction ratio of 1.0. The variant R372D was significantly different from the wild type, but not significantly different from the negative control and lost the ability to restrict.
In the suboptimal data, the negative control behaved as expected but the wild type showed a restriction ratio of 0.5 due the low infection rate. The R143D showed an intermediate phenotype statistically different from the wild type and the negative control. The variant G209S was significantly different from the wild type but not significantly different from the negative control and thus has lost the ability to restrict.
It's important to have plenty of your control cells particularly if you're new to flow cytometry. Following this or in parallel, biochemical analysis of the same mutants can be carried out to form a holistic picture.
