Cell Proliferation Kit III (EdU-FC)
Convenient, highly sensitive and non-radioactive proliferation assay for flow cytometry. Available containing different fluorophores.
One method for detection and quantification of cell proliferation is based on the incorporation of nucleoside analogs like [3H]thymidine or 5-bromo-2’-deoxyuridine (BrdU) to into the cells’ DNA during replication and visualization by autoradiography or with an anti-BrdU-antibody respectively. Both methods exhibit several limitations. Working with [3H]thymidine is troublesome because of its radioactivity and autoradiography is slow and thus not suitable for rapid high-throughput studies. The major disadvantage of BrdU staining is that the double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU units. Therefore, samples have to be subjected to harsh denaturing conditions resulting in degradation of the structure of the specimen.
The PromoKine EdU-based Cell Proliferation Kits overcome these limitations, providing a superior alternative to BrdU and [3H]thymidine assays for directly measuring DNA synthesis flow cytometry.
EdU (5-ethynyl-2’-deoxyuridine) is a nucleoside analog to thymidine and is incorporated into DNA during active DNA synthesis. In contrast to BrdU assays, the EdU-based Cell Proliferation Assays are not antibody based and therefore do not require harsh DNA denaturation for detection of the incorporated nucleoside. Instead, the EdU-based Cell Proliferation Assays utilize click chemistry for easily coupling fluorophores to the incorporated EdU – allowing fast and sensitive detection in a variety of dye fluorescent readouts. Furthermore, the streamlined detection protocol reduces the total number of steps and significantly decreases the total amount of time. The simple click chemistry detection procedure is completed within 30 minutes and is compatible with multiplexing for content and context-rich results.
Technical Library (2)
We are interested in purchasing the Live/Dead Cell Staining kit II (PK-CA707-30002) for whole cartilage tissue (diameter 3 mm punches) in combination with confocal microscopy. Does your company have experience with this application, or is there a more specific kit available?
Our Live/Dead Cell Staining Kit is for use in unfixed living cells, so it cannot be used in paraffin sections or cryosections of tissue.
There are some references reporting the use of calcein-AM and vital dyes in dissected tissues or organ cultures in vitro. The main concern would be whether the dyes can penetrate the areas of tissue that you want to examine. Also, calcein is not fixable, so you would need to observe the fluorescence in the tissue without additional processing. A longer incubation time might be required in tissue specimens compared to cells to allow the dyes to penetrate and for conversion calcein-AM into calcein. Also, because ethidium homodimer III stains cell with compromised membrane integrity, it will likely stain cells along cut edges or on the surface of a tissue slice.
A fixable alternative to calcein-AM is CFSE, which is non-fluorescent until it is hydrolyzed by esterases in the cytoplasm of living cells to generate fluorescein-SE, which covalently attaches to cellular proteins. We offer CFSE (Cell Proliferation Kit I, PK-CA707-30050), which you could pair with a vital dye like propidium iodide or ethdium homodimer III to stain dead cells. For the vital dyes, it is important to wash away the free dye thoroughly before fixing to avoid background staining.
Related Links and Documents
I have some questions about your Cell Proliferation Kit I (CFSE). 1) How long can cells survive stained? 2) What intensity is expected in the daughter cells after divisions? 3) Can readouts be performed with a general plate reader/fluorometer?
1) The Instruction Manual gives just a general labeling protocol. The length of time that you wait to image the cells or use them in flow cytometry after rinsing out the excess unreacted CFDA SE will vary depending upon the cell type. There have been reports of identifying labeled cells from a few days to 8 weeks after labeling.
2) The assay is optimized for flow cytometry or microscopy. With these methods you can directly correlate fluorescence intensity to individual cells. If you have one labeled cell that divides, the two daughter cells should each express ½ the fluorescence of the single parent cell.
The following references might be of use: – Hodgkin et al., J Exp Med 184, 277 (1996): There is an example of successive rounds of division as detected using CFDA SE in flow cytometry. – For an example of the use of CFDA SE in fluorescent microscopy: Karrer et al., Transplant Proc 24, 2820 (1992).
3) If you were to use a plate reader for a population of cells, I think that this type of correlative information would be lost and I don’t know if you would be able to glean any useful information about cell division.
Related Links and Documents
- We are interested in purchasing the Live/Dead Cell Staining kit II (PK-CA707-30002) for whole cartilage tissue (diameter 3 mm punches) in combination with confocal microscopy. Does your company have experience with this application, or is there a more specific kit available?
- Application Notes (1)
Reference Literature (3)
Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain
Burkhart et al.; Cell Mol Life Sci. 2017 Jul;74(13):2467-2485
Biochemical composition of malignant ascites determines high aggressiveness of undifferentiated ovarian tumors
Mikula-Pietrasik et al.; Med Oncol. 2016 Aug;33(8):94
Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties
Burkhart et al.; Fluids Barriers CNS. 2015 Aug 7;12(1):19
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