Flow cytometry

Also known as:

Fluorescence-activated cell sorting, FACS

*Note: Technically speaking, FACS is a derivative of flow cytometry that physically separates cells into different populations based on their characteristics. By flow cytometry it’s also possible to simply observe those different characteristics without separating the cells.

Samples needed

Single-cell suspension.

Cells will be treated in different ways depending on the measurement being made. Often, cells are immunostained for cell-surface biomarkers (i.e. proteins). Flow cytometry can be used with immunostaining for intracellular biomarkers if they are first fixed and then permeabilized to allow the antibody to enter the cells.

Controls

Recommended controls are numerous and beyond the scope of this Encyclopedia. However, they are rarely shown in published studies unless the study specifically aims to develop a new method.

Method

Figure 1.  Simplified schematic of flow cytometer being used for fluorescence-activated cell sorting (FACS). The cells in this sample have been immunostained for a cell-surface protein. This process uses an antibody that binds the cell-surface protein of interest covalently attached to a fluorescent dye. Therefore, in the image, the cells with the protein of interest are shown in red.  “Cell Sorting Using Flow Cytometry and Droplet Technology” by Sari Sabban is licensed under CC BY-SA 3.0 [Image description]

Here, we are describing the use of flow cytometry to detect cell-surface proteins. The cells are first separated to form a single-cell suspension in an appropriate buffer. Then they are immunostained for one or several cell-surface markers. The number of cell surface markers that can be detected in one experiment is limited only by the number of different wavelengths of light that can be simultaneously detected and resolved by the particular flow cytometer being used.

The cells are then allowed to pass through the flow cytometer one at a time. For each cell, two parameters called forward scatter (FSC) and side scatter (SSC) are measured, which give information about cell size and internal complexity (i.e. “granularity”), respectively. The intensity of each fluorescence signal is also measured to determine how much of each biomarker is present on the cell surface.

Interpretation

Figure 2.  Results of a flow cytometry experiment.  Relevant section of caption for published figure reads: “A, representative flow cytometry plots depicting tissue CD11b⁺Gr1⁺ recruitment 11 days after orthotopic inoculation of 4T1, 4T1 shCAIX1, and 4T1 shCAIX2 cells. B, quantification of CD11b⁺Gr1⁺ recruitment to the lungs (top) and spleen (bottom) of individual mice for each group as a proportion of the infiltrating CD45⁺ leukocyte population, n = 5 mice per group. ” “Figure 3. CAIX is required for G-CSF–mediated granulocyte recruitment to the lungs of tumor-bearing mice.” by Shawn C. Chafe et al.^[1]. [Image description]

In this experiment, the study authors have established orthotopic tumors in mice using 4T1 mouse breast cancer cells. The 4T1 cells express either a control shRNA (shNS), or one of two shRNAs against CAIX. The authors want to know how the level of CAIX expression in the tumor affects the presence in the mouse lungs of a certain subset of cells that highly express the proteins CD11b and Gr1 on their surface. To do this, the authors removed the lungs from the mice and processed them to a single-cell suspension. The cells were then immunostained for CD45, CD11b, and Gr1. CD45 is a general leukocyte marker, and Panels A and B are only showing cells from the lung that have high CD45 staining; CD45 low cells are excluded from the analysis. In the scatterplots in Panel A, each point on the graph is a cell, positioned based on the intensity of the CD11b and Gr1 staining. The researchers have divided the plot into quadrants that show the cells as “low” or “high” for each of the two biomarkers. Panel B is a dot plot that quantifies the results from Panel A by showing the overall % of the CD45⁺ cells in each sample that are CD11b⁺Gr1⁺. Based on this data, the authors conclude that when the primary tumor does not express CAIX, fewer CD11b⁺Gr1⁺ cells are found in the lung.

Image Descriptions

Figure 1 image description: Single cells passing through a funnel-shaped structure in a flow cytometer. Cells are either red to show the presence of the cell surface protein or black to show its absence. As the cells pass through the narrow part of the “funnel,” they are hit by a laser, and scattered light is detected by one of two detectors, one for forward scatter and one for side scatter. As cells come out of the “funnel” in droplets, the droplets are charged, and can be electromagnetically attracted into one of two tubes: one containing only red cells and one containing only black cells. Side panel shows a cartoon of an antibody attached to a red fluorescent dye binding to a cell-surface protein on a cell. [Return to Figure 1]

Figure 2 image description: Panel A shows three scatterplots. The % of cells (points) in each quadrant of the scatterplot are shown in the table below.

Panel B shows a dot plot comparing the % of CD45 positive cells that are CD11b⁺Gr1⁺ in the three samples. It is a quantification of the scatterplots in Panel A. The 4T1 sample shows an average of 40%, whereas both of the shCAIX samples show averages around 20%. [Return to Figure 2]

Katherine R. Mattaini
Last updated on April 3, 2022