S-phase analysis using 5-ethynyl-2 deoxyuridine (EdU) uptake
EdU like BrdU is a nucleoside analogue to thymidine which is actively transported into cells while dividing and is incorporated into the newly synthesized DNA structure. The use of antibodies with a fluorescent tag against BrdU makes it possible to determine S phase if cells were pulsed with 10uM BrdU more accurately then just using DNA binding dyes such as PI, 7-AAD, DAPI or ToPro-3. However the method by a practiced technician takes 2 hours and requires the harsh treatment of cells with 2M hydrochloric acid. Thus making it difficult to detect surface and other intracellular antigens when using the standard BrdU protocol.
S-phase analysis using bromodeoxyuridine uptake
The determination of the percentage of cells in S-phase is dependent upon the detection of a thymidine analogue, bromodeoxyuridine (BrdU), which when added to culture medium is incorporated into DNA during DNA replication and bivariate analysis of total DNA content using propidium iodide staining along the X-axis plotted against BrdU incorporation detected via FITC labelled antibodies to BrdU on the Y-axis. Immediately following a pulse of BrdU labelling, the green fluorescence (BrdU content) is observed in S-phase. If the population is sampled every 1 h to 2 h, the passage of labelled cells into G2 can be observed followed by the appearance of green fluorescence in G1 as the cells pass through mitosis. The fluorescence intensity is halved as the cells divide because the average amount of BrdU per cell is halved. Green fluorescence reappearing in S-phase is indicative of a complete cell cycle and the time taken is indicative of the cell cycle kinetics for the cell population sampled. Depending on the cell type, the BrdU pulse can be as short as 6 min and still be detectable by flow cytometry.
Although propidium iodide is probably the most commonly used dye to quantitatively assess DNA content, there are several different dyes that are available which bind stoichiometrically to DNA. These may be divided as follows:
DNA content may be measured in association with other nuclear or cellular components such as cyclins by dual fluorescence analysis in which PI/DAPI is used to stain the DNA and for example FITC or APC conjugated antibody is used to define the antigen of interest. When using DAPI most antibody fluorophores can be used except for Pacific Blue. Whilst with PI which emits at 619nm, PE, PerCP or PE-Cy5 or conjugated antibodies cannot be used as as PI emits at the same wavelength as these fluorophores.
Role of Histones in DNA Damage
DNA can be damaged by UV-irradiation and drug treatments such as, camptothecin. UV-irradiation is absorbed by pyrimidine bases, thymine and cytosine ultimately resulting in the formation of 6-4 (T-C) photoproduct, this results in a DNA damage response (DDR) causing chromatin decondensation. This results in activation of protein kinases, ATM, ATR and DNA-PKcs at the DNA damage site causing phosphorylation of p53 and histone H2AX. Hundreds to thousands of H2AX molecules are phosphorylated at Ser139 per double-stranded DNA breakage site. This phosphorylation results in the recruitment of DNA damage repair proteins to the site of the DNA damage.
Live cell cycle analysis with Hoechst 33342
Flow cytometry may be used to analyse cell cycle in live eGFP Gli2 transduced human keratinocytes, NTERTs cells by loading cells with Hoechst 33342 at 10 ug/ml for 45 mins at 37C.
Cell cycle determination by epi-fluorescence microscopy
The determination of the cell cycle analysis is a well known technique in flow cytometry. The Core Facilities at ICMS has a Metamorph epi-fluorescent deconvolution system with a software macro to determine the cell cycle stage of cells on slides or in culture.
Flow cytometry may be used to analyse cell cycle in live cells by loading cells with Hoechst 33342 at 10 ug/ml for 45 mins at 37C and PI added (5 ug/ml).
Histone H3
Histones are highly basic proteins that complex with DNA to form chromatin. H3 histone Flow cytometry may be used to analyse cell cycle with propidium iodide (PI) giving peaks of G1 and G2m with S phase located between these peaks. Labelling histone H3 with a fluorescently conjugated monoclonal antibody and then staining PI allows the investigator to determine which G2m cells are in the m phase of the cell cycle by flow cytometry.
Measurement of apoptosis by PARP
Apoptosis can be measured flow cytometrically by numerous assay's listed under Apoptosis. Cell cycle analysis can be used to measure late apoptosis by the detection of a SubG1 peak, see DNA fragmentation section. Cell cycle analysis can also be combined with the detection of cleaved PARP or Poly (ADP-ribose) polymerase-1. PARP is an 116 kDa enzyme involved DNA repair which during apoptosis is cleaved by active caspase-3. PARP can be detected flow cytometrically to show the presence of apoptosis after UV-irradiation or drug treatment.
Ploidy or endoduplication occurs when cells in G2M do not separate after DNA synthesis and proceed to synthesis DNA again become 8N cells. This can be observed in cell lines quite often with a small population of these going onto synthesis DNA again becoming 16N.
Live Go cell cycle analysis with Hoechst 33342 and Pyronin Y
Flow cytometry may be used to determine the level of quiescence or senescence in a cell population. The biological processes involved in quiescence and senescence are different in that senescence has to be induced by g-irradiation or cyclin-dependent kinase inhibitor, CDKNA1A and 2A putting cells into Go phase of the cell cycle. This cell cycle arrest after several days is irreversible and the cells grow in size without cell division and acquire SA-B-Gal staining and after a profound reorganization of chromatin structure and the endomembrane system enter a secretory state, releasing a mass of factors to the external environment called the senescence-messaging secretome (SMS) or senescence-associated secretory phenotype (SASP).
Fractional S-phase sorting using bromodeoxyuridine uptake to study gene replication timing
The determination of the percentage of cells in S-phase is dependent upon the detection of a thymidine analogue, bromodeoxyuridine (BrdU), which when added to culture medium is incorporated into DNA during DNA replication is a well known technique in flow cytometry.