Flow Cytometry and Fluorescence-Activated Cell Sorter (FACS)

00:01 Flow cytometry is a method for analyzing particles.

00:06 These could be cells or other types of particles.

00:09 In this example, we have lymphocytes incubated with a fluorescently tagged monoclonal antibody.

00:17 Perhaps we want to enumerate the number of CD4+ cells and the number of CD8+ cells in this patient.

00:27 A sheath fluid containing the antibody labeled cells passes through a small narrow tube where there is just one cell per drop. And laser light is shone onto these cells, passing through one by one down this small tube. One can measure forward light scatter from the laser light which gives a indication of the size of the cell. One can also measure 90 degree light scatter which indicates the granularity of the cell. Red and green fluorescence detectors will detect monoclonal antibodies that are binding to either CD4 or CD8. So one monoclonal antibody against CD4 might be labeled with a dye that emits green fluorescence, and the other antibody against CD8 with a dye that detects red fluorescence.

01:34 The forward scatter and side scatter allows detection of different cell types.

01:40 And we can see here an example of the type of plot that the computer linked to the flow cytometer produces.

01:47 You can see the side scatter on the Y-axis and the forward scatter on the X-axis.

01:54 And one is able to differentiate neutrophils from monocytes, from lymphocytes.

02:00 Using monoclonal antibodies, one is able to detect different populations of cells as already mentioned, for example, CD4 and CD8 T-cells.

02:10 Here we can see the CD4+ T-cells that have been stained with a monoclonal antibody coupled to the diphycoerythrin.

02:21 And the CD8+ cells detected by a monoclonal antibody coupled to the difluorescein isothiocyanate.

02:31 The cells that are not staining are mainly B-cells and NK cells because these cell types lack expression of both CD4 and CD8.

02:44 Here we see another use of this technology, detecting the double positive thymocytes.

02:50 Thymocytes are early T-cells that are developing in the thymus.

02:54 This is the only location that you ever find T-cells expressing both CD4 and CD8 because once they leave the thymus, they switch off expression of one of these two genes to become either CD4 single positive T-cells or CD8 single positive T-cells.

03:14 But here we can see very clearly on this flow cytometry plot that there are a number of cells that are staining for both CD4 and CD8; very typical of developing thymocytes, double positive, CD4+ CD8+ cells.

03:33 Flow cytometry can also be used for the detection of specific T-cells using a technique that employs peptide MHC tetramers.

03:45 Fluorescently labeled tetramers of four identical HLA molecules are loaded with particular peptides.

03:53 In this example, a peptide derived from the Epstein-Barr virus.

03:58 This allows enumeration of antigen specific T-lymphocytes.

04:05 The fluorescence-activated cell sorter is a modified version of the flow cytometer that allows the separation of different cell populations.

04:15 Here we have a mixed population of cells labeled with different fluorescent antibodies.

04:22 The mixture of cells is passed through the flow cytometer and laser light is shone onto the individual cells.

04:32 A dichroic filter allows the detection of forward scatter and also of fluorescence in different fluorescent channels depending on which particular fluorescent label is tagging the monoclonal antibody.

04:48 Also a side scatter detector can be used for analysis of the cells.

04:54 The computer that analyses all of the inputs from these different detectors and will provide a display on the computer, whereby one can see the different cells.

05:10 So here you can see some cells are double positive, in other words are tagged with both antibodies.

05:17 Some are single positive and some are completely negative.

05:22 So this is the kind of information one would get from a standard flow cytometer.

05:28 But in the fluorescence-activated cell sorter, the cells as they pass through in a single stream can be deflected in different directions depending on a charge that is put on the droplet containing the individual cell.

05:47 And as we can see here, the cells can be separated into these four different populations - ones that are positive just for one antibody binding or the other antibody binding, in other words single positive cells, the double positive cells and the double negative cells, separated into four different tubes.

06:08 One can then go on and analyze these different populations, in a variety of ways.