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Cyquant cell proliferation assay kit

Manufactured by Thermo Fisher Scientific
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The CyQUANT cell proliferation assay kit is a fluorescence-based method for quantifying the total number of cells in a sample. It utilizes a proprietary fluorescent dye that binds to cellular nucleic acids, allowing for the measurement of cell density.

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355 protocols using cyquant cell proliferation assay kit

1

Quantifying MSC DNA and Metabolism

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DNA content of MSCs was determined using CyQUANT™ Cell Proliferation Assay Kit (Life technologies™, Molecular Probes®), according to manufacturer’s instructions.
The resazurin-based assay was performed for detection of metabolic activity of MSCs. Briefly, 400 μL of fresh culture medium supplemented with 0.01 mg/mL (w/v) resazurin (Sigma–Aldrich®) was added directly to each microfluidic device. MSCs were incubated at 37 °C for 4 h, and then 100 μL from each device was transferred to a 96-well microplate. Fluorescence (λem = 530 nm, λex = 590 nm) was measured on a VICTOR™ ×3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). DNA content of MSCs was determined using CyQUANT™ Cell Proliferation Assay Kit (Life technologies™, Molecular Probes®), according to manufacturer’s instructions.
Silva D.I., Santos B.P., Leng J., Oliveira H, & Amédée J. (2017). Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform. Cell Death & Disease, 8(12), 3209.
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2

Cardiomyocyte Hypertrophy Measurement

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After 48 hours of stimulation with phenylephrine or vehicle control, NRVMs were trypsinized off of tissue culture plates, washed once in phosphate buffered saline, pelleted, snap frozen in liquid nitrogen, and stored at −80°C until analysis. Cell pellets were lysed using the CyQUANT Cell Proliferation Assay Kit (Thermo Fisher Scientific #C7026), and then sonicated. Protein concentration in the lysates was determined using a bicinchoninic acid protein assay. An aliquot of each lysate was treated with DNAse-free RNAse A (Thermo Fisher Scientific #EN0531) and the DNA concentration determined with the CyQUANT Cell Proliferation Assay Kit following the manufacturer’s instructions. NRVM hypertrophy was assessed by normalizing the protein concentration of each sample to its DNA concentration.
Garbincius J.F., Luongo T.S., Jadiya P., Hildebrand A.N., Kolmetzky D.W., Mangold A.S., Roy R., Ibetti J., Nwokedi M., Koch W.J, & Elrod J.W. (2022). Enhanced NCLX-dependent mitochondrial Ca2+ efflux attenuates pathological remodeling in heart failure. Journal of molecular and cellular cardiology, 167, 52-66.
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3

HGF Attachment and Proliferation Assay

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HGFs attachment and growth was assessed on all surfaces using CyQUANT® Cell Proliferation Assay Kit (C7026; Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions and our previous experience [24 (link)]. Briefly, in parallel cultures, 10,000 (Attachment assays) or 5000 (Proliferation studies) HGFs was seeded onto each disc and at each designated timepoint, media was removed gently with a pipette gun, followed rinse by cold PBS twice to remove unattached cells. A total of 5000 cells were seeded for proliferation to have significant surfaces area for growth as HGFs are contact inhibited. Lysates were then transferred into a new 24-well plate and stored at −80 °C. DNA content was determined by performing CyQUANT® Cell Proliferation Assay Kit (C7026; Molecular Probes, Eugene, OR, USA). Cell numbers were extrapolated using a standard curve. Three independent experiments were performed, with 6 replicates per condition per experiment. For attachment, timepoints selected were 1, 6 and 24 h, and for cell proliferation, 1, 3 and 7 days.
Li H., Guo C., Zhou Y., Sun H., Hong R, & Hamilton D.W. (2021). Titanium Substratum Roughness as a Determinant of Human Gingival Fibroblast Fibronectin and α-Smooth Muscle Actin Expression. Materials, 14(21), 6447.
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4

Mitochondrial Respiration Profiling via Seahorse XF96e

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Cells were seeded onto an XF96 cell culture microplate coated with 0.001% (w/v) poly-L-lysin (Sigma-Aldrich P4707) at 30,000 cells/well and incubated for 48 hrs in glucose-based media supplemented with 0.25 μg/ml doxycycline. On the day of measurement, cells were washed twice with Seahorse XF DMEM Basal Medium supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate and preincubated for 1 hr in a 37°C humidified CO2-free incubator. OCR was measured by a Seahorse XF96e FluxAnalyzer with the Mito Stress Test kit (Agilent). The Mito Stress Test inhibitors were injected during the measurements as follows; oligomycin (2 μM), FCCP (0.5 μM), rotenone and antimycin A (0.5 μM). The OCR values were normalized to cell density determined by the CyQUANT Cell Proliferation Assay Kit (Invitrogen C7026) according to the manufacturer’s instruction.
Senoo N., Chinthapalli D.K., Baile M.G., Golla V.K., Saha B., Ogunbona O.B., Saba J.A., Munteanu T., Valdez Y., Whited K., Chorev D., Alder N.N., May E.R., Robinson C.V, & Claypool S.M. (2023). Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant. bioRxiv.
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5

BM-MSCs Proliferation on ECM Matrices

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Human BM-MSCs from each donor were seeded at a density of 8.3 × 103 cells/cm2 on the corresponding decellularized matrices (ECMBMAd LG or ECMBMAd HG) or on plastic (control referred to as without ECM) and cultured in complete DMEM with 5.5 mM glucose. DNA level was quantified at 24, 48 and 96 h after seeding using the CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer's instructions. The DNA-bound fluorescence was measured using a Xenius XMA fluorescence microplate reader (SAFAS) at ~500 nm excitation and ~530 nm emission maxima in each lysed sample. The parameter settings were kept constant for all comparative sets of experiments. Finally, the amount of DNA was calculated from a standard curve and converted to the number of cells (one cell corresponding to 242 pg DNA).
For characterization, BM-MSCs were collected after 1 and 2 days in LG complete medium in Extract-All (Eurobio) for RNA extraction (Fig. 1C).
Entz L., Falgayrac G., Chauveau C., Pasquier G, & Lucas S. (2022). The extracellular matrix of human bone marrow adipocytes and glucose concentration differentially alter mineralization quality without impairing osteoblastogenesis. Bone Reports, 17, 101622.
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6

CyQUANT Cell Proliferation Assay for SNU308 Cells

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The cell proliferation of SNU308 cells with or without treatment was determined using CyQUANT cell proliferation assay kit (Invitrogen, Carlsbad, CA, USA).
Chiang K.C., Yeh C.N., Huang C.C., Yeh T.S., S. Pang J.H., Hsu J.T., Chen L.W., Kuo S.F., Kittaka A., Chen T.C, & Juang H.H. (2016). 25(OH)D Is Effective to Repress Human Cholangiocarcinoma Cell Growth through the Conversion of 25(OH)D to 1α,25(OH)2D3. International Journal of Molecular Sciences, 17(8), 1326.
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7

Chemotaxis Assay for Monocyte Migration

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Cellular chemotaxis assays were performed using disposable 12-well Transwell polycarbonate membrane inserts with 3-µm pore diameter (Corning). One mL of CM was plated directly into the bottom wells of the plate in triplicate. Serum-free media were plated directly into the wells as a negative control. For antibody-blocking chemotaxis assays, 2 µg/mL of human MCP-1 affinity-purified polyclonal antibody (R&D Systems), 1 µg/mL of human CCL5 monoclonal antibody (R&D Systems), 3 µg/mL human IL-6 monoclonal antibody (Clone 1936; R&D Systems), and 1 µg/mL RARRES2 MaxPab rabbit polyclonal antibody against full-length human RARRES2 (Abnova) were used for neutralization. The CM was pre-incubated with antibodies for 30 min at 37°C (5% CO2). THP-1 cells (1×106; ATCC) or human peripheral blood mononuclear cells (HPBMC; Lonza), both of which are models of human monocytes, were placed onto each chamber. The monocytes were allowed to migrate into the lower chamber for 6 hours at 37°C (5% CO2). CM was then centrifuged at 150×g for 6 minutes and the supernatants were removed. The cells were quantified by a CyQUANT cell proliferation assay kit (Invitrogen). Chemotaxis index was calculated using the following equation: Chemotaxis index = (number of monocytes in the wells of the plate − control)/total number of monocytes seeded in the top of the chamber.
Zhu Y., Tchkonia T., Stout M.B., Giorgadze N., Wang L., Li P.W., Heppelmann C.J., Bouloumié A., Jensen M.D., Bergen HR I.I.I, & Kirkland J.L. (2015). Inflammation and the Depot-Specific Secretome of Human Preadipocytes. Obesity (Silver Spring, Md.), 23(5), 989-999.
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8

Mitochondrial and Glycolytic Profiling

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PSCs or DE cells were seeded into the cell culture microplate and incubated at 37 °C incubators with 5% CO2 overnight. The DMEM assay medium (XF assay-modified DMEM supplemented with 10 mM XF glucose, 1 mM XF pyruvate, 2 mM XF glutamine, pH 7.4) was prewarmed at 37 °C to use. Then the OCR and ECAR measurement were assessed by Seahorse XF Cell Mito Stress Test Kit (Agilent, Cat# 103015-100) and Seahorse XF Glycolysis Stress Test Kit (Agilent, Cat# 103020-100), respectively. Here, 1.5 µM oligomycin (Oligo), 0.5 µM FCCP, and 0.5 µM rotenone/antimycin A (Rot/AA) were used in Mito Stress Test assay. 10 mM glucose, 1 µM oligomycin, 50 mM 2DG were used in the Glycolysis Stress Test assay. Finally, the cell number was normalized using CyQUANT™ Cell Proliferation Assay Kit (Invitrogen, Cat# C7026) or Pierce™ BCA Protein Assay Kit (Thermo Fisher, Cat# 23225).
Lv J., Yi Y., Qi Y., Yan C., Jin W., Meng L., Zhang D, & Jiang W. (2022). Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells. Cell Death Discovery, 8, 69.
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9

Evaluating ASC-Sheets on Endothelial Cell Proliferation

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To test the effect of ASC-sheets on endothelial cell proliferation, human umbilical vein endothelial cells (HUVEC, Lonza) at P4 were seeded at a density of 5000 cells/cm2 in a 96-wells plate and in a 24-wells plate and cultured overnight in endothelial growth medium (EGM-2 bullet kit, Lonza). The next day, cells were starved with 0.5% FBS in LG-DMEM for 3 h. Then, HUVEC were refreshed with either control medium (LG-DMEM 1% FBS) mixed with EGM medium (1:1) or medium conditioned by ASCs mixed with EGM medium (1:1). After 0, 1, 2, 3, and 4 days endothelial cell proliferation and viable cell numbers were determined with the Cyquant® cell proliferation assay kit (Invitrogen) and MTT assay, respectively. Combining the results from these assays will allow to (indirectly) have an indication about the proliferation. According to the manufacturers’ protocol culture plates at -80οC were frozen after removal of medium. The proliferation on each day was analyzed using known numbers of HUVEC as a DNA standard. At room temperature, 200 μl of CyQuant GR dye/lysis buffer was added to each well and incubated 5 min before reading the plate with the fluorescence microplate reader SpectraMax Gemini (Molecular Devices)
The MTT assay was based on the Mossman’s protocol [24 (link)] to check for metabolically active cells.
Sukho P., Kirpensteijn J., Hesselink J.W., van Osch G.J., Verseijden F, & Bastiaansen-Jenniskens Y.M. (2017). Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study. Stem Cell Reviews, 13(2), 267-277.
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10

Cisplatin Cytotoxicity Evaluation in CAOV3 and OVCAR3 Cells

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CAOV3 and OVCAR3 cells were seeded in a 96-well plate at a density of 5×104 cells/well, and then treated with various concentrations of cisplatin (Sinopharm) at 0, 0.25, 0.5, 1, 4, 8, and 16 µM. Twenty-four hours later, viable cells were evaluated using the CyQUANT™ Cell Proliferation Assay Kit (Invitrogen). All measured absorbances were normalized to the absorbance under control conditions.
Wang C., Qi S., Xie C., Li C., Wang P, & Liu D. (2018). Upregulation of long non-coding RNA XIST has anticancer effects on epithelial ovarian cancer cells through inverse downregulation of hsa-miR-214-3p. Journal of Gynecologic Oncology, 29(6), e99.
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