Science Focus ( Issue 21)

1 The HIV virus attacks CD4+ T cells. having more cellular components like organelles or granules). Other than cell size and complexity, we can also identify cells by proteins that are preferentially produced by the cel ls. These proteins can either be positioned on the cel l sur face or sometimes contained within the cell’s cytoplasm, like a transgenic fluorescent marker. With this in mind, as long as we are able to identify these features, we should be able to identify the cell type. For example, we can distinguish the two major types of mature T cells, helper T cells and killer T cells (a.k.a. CD4+ cells and CD8+ cells respectively), by figuring out which surface protein, CD4 or CD8, is expressed on the T cell (footnote 1). The next question, of course, is how can we identify these cellular features? Size and complexity can be measured with lasers (more on that later), but unique protein markers are harder to identify. The solution to this comes from antibodies, which are proteins that can bind specif ical ly to cer tain molecules. These antibodies can be selected such that they only bind proteins that are specific to certain cell types. These engineered antibodies are then joined (or conjugated) with unique fluorophores, which are chemical compounds that re-emits fluorescent signals at a longer wavelength upon excitation by a laser. Scientists can thus “label” the cells using these fluorophore-conjugated antibodies before the application of flow cytometry to identify the cell type. Using the example of mature T cells again, an engineered antibody-fluorophore conjugate, which can bind CD8 proteins on cell surfaces, will be able to stain CD8+ cells (killer T cells). Upon laser excitation, the fluorophore will give a unique fluorescent signal, differentiating CD8+ cells from other cell types. As discussed, scientists are able to identify specific cell types thanks to unique ma r k e r s o n t h e i r ce l l s u r face o r i n s i d e t h e i r in a tissue need to be dissociated first) is loaded into the flow cytometer. The suspension is then focused into a single stream of liquid, like how water comes out of a tap in a single stream, whereupon cel ls will be arranged into a single file before passing through an array of lasers one by one. After that, an accompanying array of sensors will detect any signals that are subsequently generated. The photons in the laser beam will be able to pass through the liquid, in which the cells are suspended in, unobstructed but photons that encounter cells in the stream will be forced to divert from their original trajectory. Such divergence of light is known as scattering. Forward scatter, which measures the amount of light diffracted slightly due to contact with the cell membrane, can be used to give a measurement of the cell’s relative size; the larger the cell, the more forward scatter there is [2, 3]. On the other hand, side scatter, which measures the amount of the l ight reflected at a greater angle upon contact with internal organelles or granules inside the cell, can give a measurement of the cell’s relative internal complexity; the more objects there are inside the cell, the more side scatter there is [2, 3]. In addition, the lasers serve to excite any fluorophores conjugated to antibodies, allowing the labeled cells to be detected. With that, cells can be identified efficiently. Besides identif ication, an extended function of flow cytometry is cell sorting. This is enabled by vibrating the stream of cells in the cytometer, causing the stream to break off into droplets containing mostly single cells. Then, the cell-containing droplets are each given different electrical charges according to their characteristics we tested before, such as the strength of fluorescence. The droplets with different charges will be deflected and sorted into different receptacles in the fluorescence-activated cell sorting (FACS) machine, so selected populations of cells, for example, successfully transformed cells that express the fluorescent marker, or tumor cells or white blood cells (including B cells and T cells) that express a specific surface protein marker [4], can be retained for analysis or further experiments. Before the invent ion of f low cytometr y, the accurate identification of specific cells from a diverse pool was almost impossible, let alone its isolation. Now, flow cytometry has become a common, yet indispensable technology in basic and cl inical laboratories. cytoplasm. W i t h t h i s background knowl edge in mind, we can now get into the procedure of flow cytometry. First, the biological sample in the form of a single-cell suspension (i.e. free- floating single cells in liquid medium; connected cells 23