Cell Count Normalization Kit

Cell Count Normalization

Enhance reliability of data on plate assay
No need for lysis procedure
Easily determine cell number just by adding the reagent

Product code

C544　 Cell Count Normalization Kit

Unit size	--
200 tests	Please inquire distributors about price.
1000 test	Please inquire distributors about price.

Component

200 tests	・Staining Solution ・Dilution Buffer ・Quenching Buffer	50 μl×1 10 ml×4 10 ml×2
1000 test	・Staining Solution ・Dilution Buffer ・Quenching Buffer	250 μl×1 100 ml×2 100 ml×1

C of A

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Product Description

Cell Count Normalization Kit employs a nucleic acid staining dye, Hoechst 33342 which binds to nuclear DNA to emit blue fluorescence. By measuring this blue fluorescence, correction of the measured value can easily be carried out in simple steps whereas the visual cell counting method requires cumbersome procedure. Moreover, unlike the correction by protein or ATP amount, the kit requires no lysis procedure. In addition, Quenching Buffer included in the kit enables a direct measuring of fluorescence signal without any background.

Manual

Technical info

When cells are analyzed in a microplate, the results obtained may sometimes differ depending on the number of cells per well. In such cases, normalization of the measured values will be necessary.

Comparison with conventional method

Normalization of the measured values at the time of plate assay is done by using the cell number or nucleus/protein level as an indicator. Among these detection techniques, our nuclear staining kit is easy to use and capable of analyzing multiple specimens.

High correlation with cell number

When you are performing a plate assay the data needs to be normalized. In order to normalize the data, an assessment method equivalent (i.e. cell counting) is essential. In the experiments below, changes in fluorescence intensity were confirmed by cell number-dependents. Data at the time of plate assay were normalized by using this kit and the cell counting method. A comparison was then made between the two methods.

Cell number-dependent changes in fluorescence intensity

Cell number-dependent changes in fluorescence intensity were observed, and the results were highly linear.

HeLa cells were serially diluted and seeded in a 96-well microplate. After incubation overnight, fluorescence intensity was measured using this kit.

Comparison with cell counting method

When normalization occurred at the time of the plate assay (lactate measurement), this kit showed equivalent results to cell counting.

2-Deoxy-D-glucose was added to HeLa cells. Lactate levels in the supernatant were quantified using the Lactate Assay Kit-WST (Item#: L256)

References

Open References

Reference No.	Normalization targets	Citation
1)	ROS Level	A. Alimu et al, "The 7-days-exposure to gammahexachlorocyclohexane causes resistance to insulin in differentiated mature 3T3-L1 adipocytes", Jpn. J. Clin. Ecol., 2020, 29(2), 45.
2)	ATP Assay Mitochondria	M. Ganbold et al, "New Amphiphilic Squalene Derivative Improves Metabolism of Adipocytes Differentiated From Diabetic Adipose-Derived Stem Cells and Prevents Excessive Lipogenesis", Front. Cell Dev. Biol., 2020, 8, 1. DOI: 10.3389/fcell.2020.577259
3)	SA-β-Gal	H. Yamamoto-Imoto et al, "Measurement of autophagy via LC3 western blotting following DNA-damage-induced senescence", STAR Protocols, 2022, 3(3), 101539. DOI: 10.1016/j.xpro.2022.101539
4)	SA-β-Gal	H. Yamamoto-Imoto et al, "Age-associated decline of MondoA drives cellular senescence through impaired autophagy and mitochondrial homeostasis", Cell Reports, 2022, 38, 110444. DOI: 10.1016/j.celrep.2022.110444
5)	SA-β-Gal	W. Klinngam et al, "Polymethoxyflavones from Kaempferia parviflora ameliorate skin aging in primary human dermal fibroblasts and ex vivo human skin", 2022, 145, 112461. DOI: 10.1016/j.biopha.2021.112461
6)	SA-β-Gal	B. Tsevegjav et al, "Holliday junction recognition protein as a prognostic biomarker and therapeutic target for oral cancer", Int. J. Oncol., 2022, 60(3), 26. DOI: 10.3892/ijo.2022.5316
7)	NAD⁺	H. Murata et al, "STAT1/3 signaling suppresses axon degeneration and neuronal cell death through regulation of NAD⁺-biosynthetic and consuming enzymes", Cell Signal., 2023, 108, 110717. DOI: 10.1016/j.cellsig.2023.110717
8)	Stem cells (585A1)	R. Abdalkader et al, "Early Differentiation Signatures in Human Induced Pluripotent Stem Cells Determined by Non-Targeted Metabolomics Analysis", Metabolites, 2023, 13(6), 706. DOI: 10.3390/metabo13060706

Q & A

Q Is it possible to measure with other fluorescent dyes at the same time?: A

If the excitation and fluorescence wavelengths overlap with those of Hoechst 33342 used in this kit, evaluation by co-staining is not possible.
Please make sure that the excitation wavelength (300-400 nm) and fluorescence wavelength (400-550 nm) of Hoechst 33342 do not overlap.

If the wavelengths do not overlap, use other fluorescence dyes for evaluation before using this kit.

Handling and storage condition

Handling and storage condition
0-5°C, Protect from light

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Cell Count Normalization Kit

Cell Count Normalization Kit