This technique can help answer key questions in the immunology field. Such as, determining how monocyte subset proportions, or expression of different markers, are altered in disease states. The main advantage of this technique, is that the cells are near to their native state, and that clear instructions and justification are used, to provide a standardized gating method.
Generally, individuals new to this method may struggle, because many papers do not give clear instructions on how gating of the cells was performed. Begin this protocol with collection of blood samples and determination of the white blood cell count, as detailed in the text protocol. Dilute the blood with phosphate-buffered saline, to adjust the concentration to approximately 5 million white blood cells per milliliter.
Prepare sufficient master mix for the number of tubes by combining blood, anti CD14-V450, anti CD16-APC and anti HLA-DR-PerCP. Vortex and pipette 51.9 microliters of mix into each tube. Add PE labeled M1 and M2 marker antibodies, as well as markers for T cells, B cells, neutrophils, and natural killer cells to their corresponding tubes.
Vortex and incubate for 30 minutes at four degrees celsius in the dark. Now, add 250 microliters of combined red blood cell lysis and white blood cell fixing solution, and immediately vortex the mixture, gently. Incubate the vortexed mixture for 10 minutes in the dark, at four degrees celsius.
After 10 minutes, add 250 microliters of PBS and spin down the cells at 260g, for 10 minutes at room temperature. Remove the supernatant, and resuspend the cells in 300 microliters of 1%formaldehyde. Store the cells at four degrees celsius, protected from light until analysis is performed.
Flow cytometry should be done within 48 hours of sample preparation. To begin, check the flow cytometer log to ensure facility staff has performed quality control checks. To set up the flow cytometry experiment, click on new experiment, followed by new specimen and new tube, to add tubes.
Select bivariate plots, by clicking on the icon, and use the dropdown menus, to select the access parameters. Ensure inclusion of a CD16/CD14 plot and a plot displaying a detector alongside time, to monitor the acquisition. Insert the tube and click, acquire.
Check the instrument voltage settings, ensuring that detector signals are not off scale. Observe cells falling in the monocyte gate of the CD14/CD16 plot. Set the recording threshold to 5, 000 events for the classical monocyte gate, and click on record.
Continue to record data for the remaining tubes. After data for all tubes has been recorded, export the flow data as fcs files for analysis. To perform monocyte gating, open files in the analysis software, double click the tube name, and select parameters from the dropdown menus to visualize the cells on a forward scatter area versus forward scatter height plot.
Create a doublet exclusion gate, by clicking on the polygon gate tool icon, and enclosing the cells. Select the gated cells by double clicking on the gated region. In the new display box, adjust dropdown menu parameters to display the cells on a forward scatter area versus side scatter area plot.
Click on the rectangular gate icon, and generously select the monocyte population, based on forward and side scatter properties, to exclude the majority of lymphocytes, natural killer cells and granulocytes. Select the gated cells and redisplay on a CD14/CD16 plot, selecting the parameters by using the dropdown menus. Click on the polygon gate, to select monocytes based on their characteristic, inverted l shape.
If the non-classical population is not distinct from the cells to its left, then contamination is likely and should be investigated. Select the gated cells, and display the monocytes on a CD/16 HLA-DR plot, by using the dropdown menus to select parameters. Click on the polygon gate to select the HLA-DR positive cells, and exclude any remaining natural killer cells and neutrophils.
Select the gated cells and display the HLA-DR positive cells on a CD14/HLA-DR plot using dropdown menus to select parameters. Click on the polygon gate, and draw a gate to exclude the HLA-DR high/CD14 low B cells. Select the gated cells and use the dropdown menus to display them on a CD16/CD14 plot.
From plot options, select zebra plot, which will enable monocyte subset gates to be drawn, to determine subset proportions. Now, click on the rectangular gate icon, and select the classical monocytes by drawing an approximate rectangular gate around the CD14 high/CD16 low classical monocyte population. Under display, select, show medians, to display the median fluorescence intensity for classical monocytes.
Adjust the gate such that the population has an equal distribution from the median on the left and on the right, and is encompassing all the cells to the left. Select the intermediate population by drawing a rectangular gate that encompasses the cells to the right of the classical gate. Adjust the bottom of the gate to exclude the non-classical cells, by aligning the gate with the bottom of the concentric circles, that are completely within the classical monocyte gate.
Gate the non-classical subset, by drawing a rectangular box down from the lower edge of the intermediate subset, selecting all the cells to the bottom of the population. Under display, select, show gate frequencies, to determine the percentage of each monocyte subset. Finally, select cells from each monocyte subset.
Alter the dropdown parameters to create a histogram for each monocyte subset, displaying each marker. Then, calculate the degree of expression, using median or geometric mean, as described in the text protocol. Successfully gated monocyte populations are shown here.
And the proportions are in line with those in the literature. The proportions for this sample, were calculated as, 88.1%classical, 4.33%intermediates and 7.49%non-classical. By assessing the relative position of other populations, it is clear that the T cells and neutrophils shown here in blue, follow well outside the monocyte, inverted l shape, on a CD16/CD14 plot.
However, both the natural killer cells and B cell populations, shown here in blue, overlapped with the non-classical monocyte population. Heat maps confirming that contaminating cell types have been removed, are shown here. The natural killer cells have been gated out by the HLA-DR CD16 step, whereas, the B cells have been gated out by the HLA-DR CD/14 step.
The expression of M1 and M2 markers in blue, relative to their isotype controls in red, are shown here. CD120b an M1 marker, shows a clear shift above its isotype control, for all monocyte subsets. This is also the case for CD93 an M2 marker, with expression being higher in classical, moderate on intermediates and low on non-classical.
While attempting this procedure, it's important to remember to ensure contaminating cells are gated out, as they can otherwise contribute to quantified expression. Following this procedure, other markers may be examined. In order to answer additional questions, such as, how monocyte marker expression changes in disease.
Don't forget that working with human blood and formaldehyde can be hazardous, and so precautions such as, personal protective equipment, and using a biohazard hood, should always be taken when performing this procedure.
