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Pathway Focus: Cell Cycle
|Organelle and Cellular Lights™ fluorescent protein-based intracellular landmarks|
Reagent delivery is mediated by an insect virus (Baculovirus) that is non-replicating in mammalian cells, thus safe to handle (Biosafety level 1). The genetically encoded and prepackaged reagents are ready for immediate use—no need to purify plasmid or worry about vector integrity and quality.
|Furthermore, no lipids, dye-loading chemicals, or other potentially harmful treatments are required. Transduction is efficient and reproducible in most cell types, including primary and stem cells—without apparent cytopathic effects:|
1. Just add the reagent to your cells for two to four hours
2. Treat with an enhancer, wash, incuabate overnight
3. Visualize your results.
These reagents open up a new avenue for studying dynamic cellular events in real space and time.
Visit www.invitrogen.com/olights to learn more about these reagents and technology, including the most up-to-date list of cell types successfully transduced with the proven BacMam technology.
|Figure 1. Montage illustrating cytoskeletal and histone dynamics during mitosis using U2-OS cells transduced with Cellular Lights™ Histone 2B-RFP and Cellular Lights™ MAP4-GFP.|
High-throughput biochemical and cell-based assays for interrogating the cell cycle signaling pathway
|LanthaScreen® kinase activity assays have been developed and validated for several key kinases in the cell cycle pathway. LanthaScreen® assays provide users the ability to:||A beta-lactamase reporter gene assay (CellSensor® assay) has also been developed and validated to monitor transcription factor E2F that turns on cell cycle responsive genes. |
Beta-lactamase provides advantages over luciferase and beta-galactosidase reporter enzymes—it can be detected in living cells with its FRET-based membrane-permeable substrate. The dual wavelength readout allows for ratiometric analysis, which significantly reduces experimental variables. Our results suggest that the CellSensor® assay can be used for the analysis of the cell cycle pathway in a high-throughput manner.
|Measurement of DNA-PK (PRKDC) activity using Lanthascreen® technology |
(A) A two-fold serial dilution of DNA-PK kinase with 1.6 mM Fluorescein-p53 [Ser15] peptide substrate and 10 µM ATP in a 384-well plate. A 2X solution consisting of 20 mM EDTA and 4 nM LanthaScreen® Tb-phospho-p53 [pSer15] Antibody was added to stop the reaction and develop the TR-FRET signal. (B) Inhibition of DNA-PK (0.45 nM) at 10 µM ATP with PI-103 (■; Literature IC50=2 nM; Observed IC50: 5 nM) and IC86621 (▲; Literature IC50=120 nM; Observed IC50: 93 nM).
|Measurement of Serum-mediated transcriptional activity of E2F using the CellSensor® dhfr(E2F)-bla NIH3T3 reporter assay.|
Left Panel: Dhfr(E2F)-bla NIH3T3 cells were plated in a 384-well format in assay medium and stimulated with Newborn Calf Serum (Invitrogen # 16010-159) in the presence of 0.5% DMSO for 15 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 3 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the 460/530 Emission Ratios plotted for unstimulated and serum stimulated samples (n=16 for each data point). Right Panel: Dhfr(E2F)-bla NIH3T3 cells were pretreated with Su9516 (EMD, #572650) at the indicated concentrations for 0.5 hours before 10% Newborn Calf Serum was added. (n=4 for each data point). IC50 of Su9516 = 0.66 µM
|Product|| Cat. No. |
|Fluorescein-p53 [Ser15] peptide substrate ||PV5132|
|LanthaScreen® Tb-phospho-p53 [pSer15] Antibody||PV5130|
|Dhfr(E2F)-bla NIH3T3 cells ||K1639|
|LiveBLAzer™-FRET B/G Substrate ||K1095|
|Live cell studies of cellular DNA content and cell cycle distribution detect variations of growth patterns due to a variety of physical, chemical, or biological means, to monitor apoptosis, and to study tumor behavior and suppressor gene mechanisms. |
In a given population, cells will be distributed among three major phases of cell cycle:
DNA content can be measured using fluorescent, DNA-selective stains that exhibit emission signals proportional to DNA mass. Flow cytometric analysis of these stained populations is then used to produce a frequency histogram that reveals the various phases of the cell cycle.
|Molecular Probes® offers a stain with near-infrared emission for DNA content analysis in living cells—Vybrant® DyeCycle™ Ruby.|
Vybrant® DyeCycle™ Ruby is cell membrane-permeant, DNA-selective, and essentially non-fluorescent until bound to double-stranded DNA. This dye takes advantage of the commonly available 488 nm and 633/5 nm excitation sources with emission >670 nm, placing cell cycle studies within reach of all flow cytometrists while leaving common blue laser channels open for other studies (Figure 1). This dye can penetrate the membranes of living cells and bind to DNA without killing the cells. Now researchers can assess cell proliferation and then use those same cells post-sorting for further studies.
Learn more about the use of Vybrant® DyeCycle™ Ruby Stain
|Product|| Cat. No. ||Size|
|Vybrant® DyeCycle™Ruby Stain ||V10309||100 Assays|
|Vybrant® DyeCycle™Ruby Stain ||V10273||400 Assays|
|Cell proliferation and the characterization of agents that either promote or retard cell proliferation are extremely important areas of cell biology and drug-discovery research.|
The succinimidyl ester of carboxyfluorescein diacetate (5(6)-CFDA, SE or CFSE, C1157) is currently the most widely used probe for generation analysis of cells. CFDA SE spontaneously and irreversibly couples to both intracellular and cell-surface proteins by reaction with lysine side chains and other available amine groups.
When cells divide, CFDA SE labeling is distributed equally between the daughter cells, which are therefore half as fluorescent as the parents. As a result, each successive generation in a population of proliferating cells is marked by a halving of cellular fluorescence intensity that is readily detected by any conventional flow cytometer.
|CFDA SE produces more homogenous cellular labeling and, consequently, much better intergenerational resolution than other cell-tracking dyes, such as the membrane marker PKH26. Using flow cytometric analysis of CFDA SE labeling, researchers can reliably resolve 8 to 10 successive generations of lymphocytes (figure 1) CFDA SE labeling is also useful in tracing of populations in vivo up to several weeks.|
|Product|| Cat. No. |
|CellTrace™ CFSE Cell Proliferation Kit - for flow cytometry ||C34554|
Click-iT® EdU Cell Proliferation Assays - Improve upon traditional methods for detecting and quantitating newly synthesized DNA.
|Like 3H-thymidine and BrdU-based assays, the Click-iT® EdU assay is based on detecting the incorporation of a nucleoside analog into newly synthesized DNA, but in this case, the nucleoside analog is EdU (5-ethynyl-2’-deoxyuridine), and it is detected not with an antibody but by a click reaction–a copper-catalyzed covalent reaction between an azide and an alkyne. In the Click-iT® EdU assay, the EdU nucleoside contains the alkyne and the fluorescent detection reagent contains the azide. |
In contrast to the BrdU detection, the Click-iT® EdU detection protocol does not require a harsh DNA denaturation step to allow the detection reagent access to the incorporated nucleoside.
Learn more about Click-iT EdU at www.invitrogen.com/click
|Figure 1—Detection of the incorporated EdU with the Alexa Fluor® azide versus incorporated BrdU with an anti-BrdU antibody. The small size of the Alexa Fluor® azide eliminates the need to denature the DNA in order for the detection reagent to gain access to the nucleoside.|