Immunology Fall 2026: Lecture 14 Flow Cytometry
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Overview
Brianne Barker explains how flow cytometry uses fluorescently labeled antibodies and laser-based measurements to identify and quantify specific cells in heterogeneous samples. She covers CD marker naming, forward and side scatter, fluorescence plots, sequential gating, and how fluorescence-activated cell sorting (FACS) physically recovers selected cells.
Key takeaways
- Fluorescent antibodies turn cell-surface proteins such as CD3 and CD8 into measurable labels, letting researchers identify cell types that microscopy can only classify broadly.
- Forward scatter estimates cell size, while side scatter estimates granularity or internal complexity; together, these measurements separate major blood-cell populations.
- Every event in a flow cytometry dot plot represents an individual cell, enabling rapid analysis of samples containing roughly 100,000 cells.
- Gating converts plots into quantitative subsets and supports stepwise analysis, such as isolating lymphocytes, identifying CD3-positive CD8-positive cells, and measuring CD45RA.
- Analytical flow cytometry typically sends measured cells to waste, whereas FACS uses charged droplets and deflection plates to physically collect selected cells.
Chapters
- Flow cytometry is a core immunology technique for reading data about cell populations throughout the course.
- A Moodle problem set on flow cytometry is assigned alongside the lecture, with time provided for questions at office hours.
- The central challenge is identifying and quantifying particular cells within a heterogeneous mixture.
- Antibodies detect biomolecules by binding specifically to their epitopes, rather than by performing immune functions such as neutralization or opsonization.
- Antibody-based methods include blood-typing agglutination, pregnancy and rapid COVID tests, ELISAs, Western blots, immunofluorescence, and flow cytometry.
- Different antibody assays vary in sensitivity, meaning how much antigen is needed to generate a detectable signal.
- A milliliter of blood contains about 5 million red blood cells, making rare leukocytes difficult to locate and count manually.
- Microscopy may distinguish broad cell types, but lymphocytes include CD4 T cells, CD8 T cells, B cells, and other populations that can look similar.
- Tracking HIV-specific CD8 T-cell responses in mouse blood requires identifying cells far more precisely than visual inspection allows.
- Without standardized names, different researchers may give the same cell-surface protein multiple names, complicating literature searches and comparisons.
- The CD system, short for cluster of differentiation, assigns standardized names such as CD1, CD2, and CD3 to leukocyte surface proteins.
- Some markers have lettered subtypes, including CD1A through CD1E, and familiar alternative names may remain in use.
- Researchers select antibodies against characteristic cell-surface proteins, such as CD3 on T cells and CD8 on cytotoxic T cells.
- Antibodies are commonly attached to fluorescent tags at the Fc region, allowing a detector to identify cells carrying each marker.
- A cell labeled by both anti-CD3 and anti-CD8 is identified as a CD8 T cell, unlike cells expressing only one or neither marker.
- Fluorescent antibodies identify cells, but inspecting 100,000 labeled cells by microscope would remain slow and prone to missed cells.
- A flow cytometer measures individual cells as they pass through the instrument, automating the readout for large samples.
- The workflow starts by labeling a heterogeneous cell mixture with antibodies, then placing the sample in the cytometer.
- The instrument suspends cells in saline or sheath fluid and uses pressure to focus the sample stream into single-file flow.
- Hydrodynamic focusing presents individual cells to the laser for separate measurements, with the system recording data for each cell.
- In standard analytical flow cytometry, measured cells proceed to waste rather than being recovered.
- Forward scatter (FSC) measures low-angle scattered light and serves as an approximate indicator of cell size.
- Side scatter (SSC), typically detected at 90 degrees, reflects internal complexity and granularity, such as the granules in granulocytes.
- Plotting FSC against SSC can distinguish small, low-complexity lymphocytes from larger monocytes and highly granular neutrophils.
- Each dot in a flow cytometry scatter plot represents one cell; Barker’s PhD example includes roughly 100,000 measurements collected in about two minutes.
- Contour plots emphasize regions with many cells, while pseudocolor plots show both individual events and density, with dense areas rendered red and sparse areas blue.
- Two-dimensional FSC and SSC plots reveal distinct populations that can be obscured in separate one-dimensional histograms.
- A fluorophore emits light at a characteristic wavelength after excitation by laser light, and optical filters direct emissions to the corresponding detectors.
- Cytometers vary in capacity: an example instrument had four fluorescence detectors plus FSC and SSC, while later machines could measure 16 colors or more.
- A two-color plot separates cells into four groups: positive for both markers, positive for either marker alone, or negative for both.
- Gating draws regions around cell populations to calculate their proportions; one example separates lymphocytes at 60.7%, monocytes at 3.81%, and granulocytes at 28.4%.
- Quadrant gating divides a two-marker plot into positive and negative groups, and gated cells can be analyzed again using additional markers.
- Sequential gates can isolate lymphocytes, then CD3-positive CD8-positive cells, and then examine markers such as CD45RA.
- A thymus example shows CD4/CD8 quadrants with about 80% double-positive cells, 12% CD4-positive single-positive cells, 5% CD8-positive single-positive cells, and 3% double-negative cells.
- Barker’s mouse study gated lymphocytes and T cells, then measured HIV-specific CD8 T cells; one sample contained 3.05% cells matching the target markers.
- In FACS, charged droplets are deflected between charged plates into collection tubes, allowing researchers to recover a purified population for further experiments.
Transcript excerpt
0:00 Hi everyone. During the lecture on October 7th, some of the videos that I was showing in class forced my recording to stop unexpectedly. So, the first half of this lecture is recorded during class on October 7th, and then you'll see that once the videos some of the other videos start, um it switches to something that I recorded after the fact. Sorry about that, but hope this is helpful. All right. Hi everybody. Um I'm going to go ahead and get started. So, I know that the first question that always comes up is about whether or not I have everything graded, and no I don't. My usual goal for things for exams is a week. Um
0:48 so, we're going to push to have it for Friday, um but we'll see what the deal is. Um other things just to be aware of, um as you can see today we're going to be talking about flow cytometry, um which I moved from um before the other exam. Um this is a really critical technique in immunology that immunologists use all the time, and um you're certainly going to be seeing data and sort of thinking about uh flow cytometry data um throughout the rest of the semester. Um in fact, some of you saw some flow cytometry um plots on the old practice exams, um but we hadn't talked about that yet. Um because it's sort of really important
1:35 that you have the ability to kind of read and understand flow cytometry data throughout the rest of the course, I have an assignment uh problem set about flow cytometry um that is posted on Moodle. Because we're talking about flow cytometry now, I wanted to give you a week for that problem set, so it's due on Wednesday,
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Related notes
Summary, takeaways, and chapters were generated by AI from the video's transcript and may contain errors. The video belongs to its creator, Brianne Barker.