Cell Cycle Analysis Tritiated (3H) Thymidine uptake was one of the first methods used to assess cell division

Colorimetric Assays for Cell Division Scientists developed a number of different assays in which metabolically active cells cleave colorless substrates into colored, often insoluble products that can then be measured spectrophotometrically. In one such assay, the tetrazolium compound MTT (3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow colored compound is reduced by metabolically active cells to form insoluble, purple formazan dye crystals. The absorbance of each cell sample can then be read directly in the culture wells at 570 nm, the absorbance peak of formazan.

Colorimetric Assays for Cell Division The more metabolically active cells that are present in the culture, the more formazan will be generated, and so this assay provides a readout of the number of live cells as a function of time. In this way, the MTT assay can measure cell proliferation or cell death.

Bromodeoxyuridine-Based Assays for Cell Division Use Antibodies to Detect Newly Synthesized DNA Bromodeoxyuridine (BrdU) is a Thymidine analogue. When introduced into cells, BrdU is rapidly phosphorylated to bromodeoxyuridyl triphosphate (an analogue for deoxythymidine triphosphate) and is incorporated in its place into newly synthesized DNA. Cells that divide following BrdU incorporation can then be identified using antibodies to BrdU. In recent years, other chemical analogues that mimic BrdU’s functions as a marker of dividing cells have been generated, including EdU, which can be detected using specific reagents.

NES143 monolayer cells; CY3-BrdU

Propidium Iodide Enables Analysis of the Cell Cycle Status of Cell Populations Propidium iodide is a fluorescent dye with a flat, planar structure that slides between the rungs of (or intercalates into) the DNA ladder in a quantitative manner. By using the flow cytometer to measure the fluorescence from a population of propidium iodide-labeled cells, we can identify which cells within the populations are at each stage of the cell cycle. G1 cells will have half the DNA of G2 cells, or cells about to undergo mitosis, and cells that are currently replicating DNA and are therefore in S phase will have an intermediate value. Apoptotic cells and fragments that have begun to break down their DNA will appear as events with less than G1 amounts of DNA. Other dyes that bind to DNA and allow for similar types of cell cycle analysis include DAPI, Hoechst 33342, and 7-amino-actinomycin D (AAD).

Carboxyfluorescein Succinimidyl Ester Can Be Used to Follow Cell Division Carboxyfluorescein succinimidyl ester (CFSE), also known as carboxyfluorescein diacetate succinimidyl ester (CFDASE) The diacetyl groups enable the CFDASE to enter the cell, and are then cleaved by intracellular esterases, so that the CFSE remains trapped within the cytoplasm. In the cytoplasm, molecules of CFSE are efficiently and covalently attached to intracytoplasmic proteins. Attachment of CFSE to intracytoplasmic proteins occurs with essentially no deleterious effects on cellular metabolism or cell division.

Carboxyfluorescein Succinimidyl Ester Can Be Used to Follow Cell Division The power of CFSE labeling lies in the fact that the amount of fluorescence emitted is cut in half each time the cell divides. The right-hand peak represents those cells that did not divide after CFSE incorporation. The peak to its immediate left represents cells that have divided once, the next one to the left represents cells that have divided twice, and so on. One can use the sorting capacity of the flow cytometer to physically separate those cells that have not divided, or have divided once, twice, or more times, and then analyze the separate cell populations for the expression of particular genes.

Assays of Cell Death

The 51Cr Release Assay Was the First Assay Used to Measure Cell Death Target cells are first incubated in a solution of sodium 51Chromate, which is taken up into the cells. Excess chromium is washed out of the cell suspension and the radioactively labeled targets are mixed with the killer cell population at defined effector to target cell ratios. Death of the target cells is indicated by the release of 51Cr into the supernatant of the mixed cell culture, and is quantified by comparing the 51Cr release from the test cells with that from detergent treated and control cells. A modern alternative to this technique uses CFSE to label the cells and quantifies the release of fluorescent material into the supernatant with a fluorescent plate reader.

Fluorescently Labeled Annexin V Measures Phosphatidyl Serine in the Outer Lipid Envelope of Apoptotic Cells In cells undergoing apoptosis (programmed cell death), but not other modes of cell death, the membrane phospholipid phosphatidyl serine flips from the interior to the exterior side of the plasma membrane phospholipid bilayer. Phosphatidylserine is translocated to the external membrane and serves as a recognition signal for phagocytes. Annexin V is a protein that binds to phosphatidyl serine in a calcium-dependent manner. Fluorescently labeled Annexin V can therefore be used to tag apoptotic cells for detection using flow cytometry or a fluorescent plate reader.

The TUNEL Assay Measures Apoptotically Generated DNA Fragmentation Apoptotic cells undergo a process of progressive DNA degradation, which results in the generation of short DNA fragments within the nucleus. The TUNEL assay relies on the use of the enzyme terminal deoxyribonucleotidyl transferase (TdT) to add bases onto the broken ends of DNA sequences in a non-templated manner. The classic variation of the TUNEL method uses TdT to add BrdU to fixed and permeabilized cells. BrdU is incorporated into the newly synthesized DNA, and is then detected with fluorescently labeled anti-BrdU antibodies.

More recent varations of this method use the incorporation of short DNA segments prelabeled with a molecule that then binds to a fluorescent tag under very gentle conditions.

Caspase Assays Measure the Activity of Enzymes Involved in Apoptosis The caspases are a family of cysteine proteases that cleave proteins after aspartic acid residues. Different members of the caspase family are activated during apoptotic cascades, depending on their mode of initiation. For example, caspase 8 is activated upon engagement of the Fas receptor by Fas ligand. Several different types of caspase assays are now commercially available, including caspase detection kits that yield yield fluorescent products upon caspase-mediated cleavage, as well as kits that detect the cleaved and active forms of caspases using Western blot methodology.