The BrdU Cell Proliferation Assay Bundle detects 5-bromo-2’-deoxyuridine (BrdU) built-in into cell DNA all through cell proliferation using an anti-BrdU antibody. When cells are cultured with labeling medium that includes BrdU, this pyrimidine analog is built-in as a substitute of thymidine into the newly synthesized DNA of proliferating cells.
After eradicating labeling medium, cells are mounted and the DNA is denatured with our fixing/denaturing reply. Then a BrdU mouse mAb is added to detect the built-in BrdU (The denaturing of DNA is crucial to reinforce the accessibility of the built-in BrdU to the detection antibody). Anti-mouse IgG, HRP-linked antibody is then used to acknowledge the sure detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of BrdU built-in into cells, which is a direct indication of cell proliferation.
Product Consists of
Quantity (with Rely)
Reply Color
BrdU
1 x 150 µl
Fixing/denaturing Reply
2 x 25 ml
BrdU Mouse Detection mAb
1 x 500 µl
Inexperienced
Anti-mouse IgG, HRP-Linked Antibody
1 x 500 µl
Pink
Detection Antibody Diluent
1 x 50 ml
Inexperienced
HRP Diluent
1 x 50 ml
Pink
20X Wash Buffer
1 x 50 ml
TMB Substrate 7004
1 x 50 ml
STOP Reply 7002
2 x 25 ml
Background
Halogenated nucleotides such as a result of the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in residing cells and tissues. BrdU turns into built-in into replicating DNA as a substitute of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies permits labeling of cells in S a part of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb could be utilized to detect BrdU built-in into single stranded DNA.
Brdu Cell
Key choices and particulars
Sensitivity: 40 cells/successfully
Sample variety: Adherent cells, Suspension cells
Detection approach: Colorimetric
Assay variety: Sandwich (qualitative)
5-bromo-2-deoxyuridine (BrdU) is a pyrimidine analog. It would get built-in into the newly synthesized DNA of proliferating cells as a substitute of thymidine. Biovision’s BrdU Cell Proliferation Assay Bundle detects built-in BrdU using a mouse anti-BrdU antibody. An anti-mouse HRP-linked secondary antibody is used to detect the anti-BrdU antibody sure to BrdU, which is adopted by addition of TMB (a HRP substrate). The extent of color enchancment is proportional to the quantity of BrdU built-in into the cells and could be utilized straight as an indicator of cell proliferation. Compared with totally different cell proliferation assays, this bundle detects solely the proliferating cells and by no means the seeded cells. This extraordinarily delicate, non-radioactive bundle detects as a lot much less as 50-100 proliferating cells.
METABOLIC PROLIFERATION ASSAYS
Assays that measure metabolic train are acceptable for analyzing proliferation, viability, and cytotoxicity. The low cost of tetrazolium salts much like MTT, XTT, and WST-1 to colored formazan compounds or the bioreduction of resazurin occurs solely in metabolically energetic cells. Actively proliferating cells improve their metabolic train, whereas cells uncovered to toxins might have decreased train.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue) is a water soluble tetrazolium salt yielding a yellowish reply when prepared in media or salt choices lacking phenol pink. Dissolved MTT is remodeled to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes. This water-insoluble formazan shall be solubilized using isopropanol or totally different solvents, and the dissolved supplies is measured spectrophotometrically using absorbance as a function of focus of remodeled dye.
XTT Cell Proliferation Assays
In distinction to MTT, the cleavage product of XTT is soluble in water; a solubilization step is subsequently not required. The tetrazolium salt XTT is cleaved to formazan by a elaborate cell mechanism. This bioreduction occurs in viable cells solely, and is expounded to NAD(P)H manufacturing by way of glycolysis. The amount of formazan dye long-established straight correlates to the number of metabolically energetic cells inside the custom.
WST-1 Cell Proliferation Assays
The regular tetrazolium salt WST-1 is cleaved to a soluble formazan by a elaborate cell mechanism that occurs totally on the cell ground. This bioreduction is principally relying on the glycolytic manufacturing of NAD(P)H in viable cells. The amount of formazan dye long-established straight correlates to the number of metabolically energetic cells inside the custom.
Description: Cell Biolabs? CytoSelect Cell Proliferation Assay Reagent (Fluorometric) provides a fluorometric format for measuring and monitoring cell proliferation. Cells can be plated and then treated with compounds or agents that affect proliferation. Cells are then incubated with the proliferation reagent. Upon entering metabolically active live cells, the non-fluorescent proliferation reagent is converted into a bright red fluorescent form. An increase in cell proliferation is accompanied by increased fluorescent signal, while a decrease in cell proliferation (and signal) can indicate the toxic effects of compounds or suboptimal culture conditions. The assay principles are basic and can be applied to most eukaryotic cell lines, including adherent and non-adherent cells and certain tissues. This cell proliferation reagent can be used to detect proliferation in bacteria, yeast, fungi, protozoa as well as cultured mammalian and piscine cells. The kit contains sufficient reagents for the evaluation of 960 assays in ten 96-well plates or 192 assays in eight 24-well plates.
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