Cell Proliferation Kit I (CFSE)
For flow cytometry or microscopy
The PromoKine Cell Proliferation Kit I (CFSE) provides a convenient and sensitive way to determine cell proliferation or cell division. The assay is based on the amine-reactive dye CFDA SE (5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester; also known as CFSE) which is non-fluorescent and can diffuse passively into cells. When it enters the cells, the acetate group is removed by intracellular esterases yielding the bright green-fluorescent dye carboxyfluorescein SE. Its succinimidyl ester (SE) reactive group reacts with intracellular amines and attaches the fluorescent dye to intracellular proteins, retaining it in the cells and allowing fixation with aldehyde fixatives. CFDA SE has been used as a tracer for several cell divisions as the label is stably inherited by daughter cells through successive cell divisions or after cell fusion and is not transferred to adjacent cells in a population.
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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