Illustrating Flow Cytometry in an Immunology Lab by Using a Hands-On, Low-Tech Simulation

INTRODUCTION

Flow cytometry is a foundational technique in immunology, microbiology, and other cell and molecular biology applications. The basic method involves passing suspended cells through a flow cytometer, which can detect and quantify the forward and side scattering of light passing through the cells. The cells can also be incubated with fluorescent probes or fluorophore-conjugated antibodies prior to passing them through the flow cytometer, allowing the presence and relative expression level of specific molecules to be analyzed. Cells can also be gated for the expression of specific molecules, and further analysis of other labeled molecules in the gated subpopulations of cells can then be performed. The data from a flow cytometry experiment is typically shown as dot/scatter plots or histograms. The cost of flow cytometry equipment has decreased substantially over the past few decades. However, it is still a major equipment purchase that is not available at many primarily undergraduate institutions, where flow cytometry-based experiments might typically be done in the context of a microbiology or immunology course rather than as a mainstay of a major research project. The cost of fluorescent-tagged antibodies and the availability of cells to label for a lab activity can also be barriers to doing flow cytometry experiments in an undergraduate lab course.

In our Immunology course, students see flow cytometry data in their textbook (1) and in selected primary literature articles presented by the students in a journal club format in some of the lab sessions for the course. We have found over many years that students often struggle to understand how to interpret flow cytometry data. A recent article by Fuller et al. described an active learning activity in which students analyzed raw flow cytometry data with FlowJo software and showed gains in student confidence in flow cytometry data interpretation and gating strategies (2). We do not have access to a flow cytometer on our campus to give students firsthand experience with this technique or to generate raw data for them to analyze, nor do we have the necessary software for analysis. Instead, we have developed a low-cost, low-tech simulation using rubber bouncy balls of different mixed color patterns to represent the individual cells passing through the flow cytometer.

PROCEDURE

This activity was designed for a 3-hour lab period with up to 20 advanced undergraduate students working in five groups of three or four students per group. The detailed handout that students were given is available in Appendix 1. This activity was performed about 4 weeks into the semester after students had been exposed to a brief student-driven techniques presentation on flow cytometry and interpreted flow data in a minimum of one primary research paper. Briefly, each group of students was given a bucket containing a random sample of 50 to 60 bouncy balls (we purchased the Fun Central brand 27-mm bouncy ball bulk pack) that had a variety of color combinations. Ten non-ball objects such as caps from screwcap tubes were included to represent red blood cells or cell debris. Students were instructed to blindly take one ball (or non-ball object) out of the bucket at a time to represent a cell/object moving through the fluid stream past the lasers and detectors in the flow cytometer. For the first activity, students estimated the amount of white color on each ball versus the amount of dark colors such as dark green or dark blue to represent forward scatter and side scatter, respectively. Students hand-plotted their results on grid paper to create a dot plot of their “cell” sample. We chose to require students to create their plots by hand for all of the activities because 1) certain types of plots are not easy to create in the software available on our lab computers (Excel), and 2) to reinforce the fact that each data point represented an individual cell with its quantified characteristics as determined on the basis of the color pattern on the ball.

For each of the remaining activities, students put all of the balls back in their bucket and again pulled them out one ball at a time. For the second activity, they were instructed to use the proportion of white on each ball to represent staining for CD11c, a marker for myeloid cells including dendritic cells. For this activity, data were plotted in histogram form, with the number of cells on the y axis and the percentage of white color on the ball on the x axis. For the third activity, student groups had to use their textbook and internet resources to determine an appropriate marker for different T cell subsets and assign the different colors on the balls to each to represent a specific T cell marker, such as CD8. This represented a sample of cells stained with multiple fluorescent-tagged antibodies, allowing sorting of cells into different T cell subsets. Students plotted their data points on three different scatter/dot plots representing cytotoxic T cells versus helper T cells, T_H1 versus T_H2 cells, or T_FH versus T_reg cells. Examples of student hand-plotted graphs can be found in Appendix 2. The plots generated by each student group were submitted at the end of the lab session for grading. After completing the lab activity, students were administered a voluntary opinion survey. This study was approved by the Missouri Western State University Institutional Review Board (IRB), proposal ID #783. The results of this survey submitted by 33 students from two course offerings are presented in Table 1. Students indicate a lack of understanding of flow cytometry and lack of confidence interpreting flow data prior to the activity. Following the activity, a high percentage of students agree and strongly agree the activity helped them learn and help them gain confidence in understanding flow data plots.

TABLE 1.

Student survey data.

	Agree and Strongly Agree	Neutral	Disagree and Strongly Disagree
Before doing the flow cytometry lab activity, I understood the technique of flow cytometry.	12.1%	39.4%	48.4%
The flow cytometry simulation lab activity helped me better understand the technique of flow cytometry.	100%	0%	0%
The flow cytometry simulation lab activity helped me understand the flow cytometry data figures in the journal articles we read later in the semester.	91%	6%	3%
The flow cytometry simulation lab activity was not a good use of lab time.	0%	0%	100%
The flow cytometry simulation lab activity helped me better understand the different ways in which flow cytometry data is presented (histograms, dot plots, etc.).	75.8%	21.2%	3%
Before the flow cytometry lab activity, I was confident in interpreting flow cytometry data plots.	6%	24.2%	69.7%
I am confident that I can now accurately interpret flow cytometry data presented as a histogram.	78.8%	18.2%	3%
I am confident that I can now accurately interpret flow cytometry data presented as a dot plot.	87.9%	12.1%	0%
I am confident that I can now accurately interpret flow cytometry data presented as a contour plot.	63.6%	36.4%	0%
The flow cytometry student presentation was sufficient for me to understand how flow cytometry works.	54.5%	24.2%	21.2%

CONCLUSION

The vast majority of students were actively engaged with their groups during the activity. In a few cases, some groups had to spend a few minutes digesting the information and going through the instructions. This led to a small number of comments on the student survey indicating confusion regarding the instructions or a desire to have more direction. In this open response section of the student survey, we found several common themes. More than half the students commented that this activity helped them better understand the functioning of a flow cytometer and its power to generate a massive number of individual data points. Similarly, several students commented that plotting the data helped them understand histograms and dot plots, the two plot types we asked them to generate. Besides the small number of comments about wishing for additional or clarified instructions, five students commented the activity was too time-consuming and redundant. A surprising number (30%) commented they were uncomfortable making judgments, individually or in their groups, about the percentage of a given color on the surface of the ball. A variation to this activity, suggested by a few student comments, would be to incorporate balls of various sizes into the mix, especially during the first part of the activity simulating cell sorting by forward and side scatter.

In summary, this activity allows undergraduate students to simulate flow cytometry in an immunology course when an instrument is not available. Student survey data and comments overwhelmingly indicate this activity helps undergraduates understand the basic workings of a flow cytometer, how flow data can be plotted, and how to interpret those data plots. We find this activity to be a productive use of time to aid student learning. It can be performed in parts or as a whole, in either a lab setting or lecture classroom setting.