Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry

Our research identifies microglia and infiltrating macrophages in spinal cord injury to clarify their roles in neuroinflammation using optimized flow cytometry. Flow cytometry and density gradient centrifugation are key for isolating and phenotyping central nervous system immune cells with high specificity and efficiency. Distinguish morphologically similar microglia and macrophages, minimizing cell loss during isolation, and inferior microspecificity in dynamic injury microenvironments remain key hurdles. Existing methods fail to reliably separate resident microglia from infiltrating macrophages. Our marker-based approach resolves this critical limitation in spinal cord injury studies.

[Presenter] To begin, analyze the isotype control tube using the flow cytometer with the compatible software. Adjust the forward scatter and side scatter voltages to position the cell population within an appropriate range. Set up two additional graphs, one with IgG APC on the X-axis and IgG PE on the Y-axis, another with IgG-FITC on the X-axis, and IgG APC-Cy7 on the Y-axis. Then adjust the fluorescence channel voltages for IgG APC, IgG PE, IgG FITC, and IgG APC-Cy7 to position the cell population within the 10 to the power of three of the double-negative region. After confirming the optimized voltages using the isotype control tube, retain all settings. Proceed to analyze the antibody-stained experimental tubes. Establish a graph with CD11b PE on the X-axis and side scatter on the Y-axis. Set up another graph with CD68 FITC and CCR7 APC-Cy7. Using the isotype control as a reference, delineate the CD11b-positive region and define it as the P1 gate. Select the CD68 FITC and CCR7 APC-Cy7 graph. Right click to open Show Population, and assign the F4 column to P1. After acquiring the data, adjust the fluorescence compensation as needed. Analyze and record the percentage of CD11b CD68 CCR7-positive and CD11b CD68-positive CCR7-negative cell populations. Begin analyzing the experimental tubes using the voltage settings from the isotype control tube. Establish a graph with CD45 APC and CD11b PE. Then set up two graphs with CD68 FITC and CCR7 APC-Cy7. Acquire 50,000 events per sample and adjust the fluorescence compensation after data collection. In the pseudocolor plots of CD45 and CD11b, identify and analyze the regions for CD11b-positive, CD45-negative low, and CD11b-positive CD45 high cells. Then analyze the CD68-positive CCR7-positive and CD68-positive CCR7-negative populations within each region. Integrate all analyses to determine the percentage of M1-like and M2-like microglia and macrophages across the defined regions. Launch the flow cytometry analysis software. Drag the flow cytometry experiment files into the All Samples section of the interface. Double-click on the isotype control sample to open a two-dimensional dot plot. Select FSCA for the X-axis and SSCA for the Y-axis, and click on the rectangular gate button and use it to select the area excluding cellular debris. Double-click the gated area to open a new histogram plot. Then select FITC for the X-axis and histogram for the Y-axis. Click on the region gate button to define the threshold between negative and positive fluorescent signals. For each of the four fluorescent antibodies, determine the threshold between negative and positive signals using the isotype control sample. Sequentially change the X-axis fluorochrome to PE-APC and APC-Cy7 while keeping the Y-axis's histogram. Use the region gate tool in each histogram to finalize gates for distinguishing negative and positive fluorescent signals for the four antibodies. Next, double-click on the experimental sample to open a dot plot. Select FSCA for the X-axis and SSCA for the Y-axis, Click the rectangular gate button to gate the population after removing debris. Double-click the gated region to open a new dot plot. Select CD11b PE for the X-axis and SSCA for the Y-axis. Click the rectangular gate button and use the PE threshold from the isotype control to define the CD11b-positive gate. Now double-click the CD11b-positive region to open another dot plot, Select CD68 FITC for the X-axis and CCR7 APC-Cy7 for the Y-axis. Click the crossgate button and use the isotype-derived FITC and APC-Cy7 thresholds to categorize cells into M1-like and M2-like types. The proportion of CD45 high CD11b-positive CD68-positive CCR7-positive cells was significantly increased in the spinal cord injury, or SCI, vehicle group compared to the sham group, and CRID3 treatment did not alter this level. The proportion of CD45-negative low CD11b-positive CD68-positive CCR7-positive cells was significantly higher in the SCI vehicle group compared to sham, and was significantly reduced following CRID3 treatment. The proportions of CD11b-positive CD68-positive CCR7-positive cells were significantly increased in the SCI vehicle group compared to sham and significantly decreased after CRID3 treatment. CD11b-positive CD68-positive CCR7-negative and CD45-negative low CD11b-positive CD68-positive CCR7-negative cell proportions were significantly decreased in both SCI vehicle groups compared to sham and increased after CRID3 treatment.