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Brdu incorporation assay

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The BrdU incorporation assay is a laboratory technique used to measure cell proliferation. It detects the incorporation of the synthetic thymidine analog bromodeoxyuridine (BrdU) into the DNA of dividing cells, which indicates active DNA synthesis and cell division. The assay provides a quantitative measure of cellular proliferation without making any interpretations about the intended use of the technique.

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Lab products found in correlation

Infinite 200 pro plate reader, by Tecan (2 mentions) Anti cd3 antibody 145 2c11, by BioLegend (2 mentions) Multscango, by Thermo Fisher Scientific (1 mentions) Recombinant il 1β, by BioLegend (1 mentions) Leaf purified anti mouse ifn γ antibody, by BioLegend (1 mentions) Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions) Tunel assay, by Abcam (1 mentions) Infinite m200, by Tecan (1 mentions) Mtt solution, by Merck Group (1 mentions) Axioskop 2 plus, by Zeiss (1 mentions) Ab6741, by Abcam (1 mentions)

Spelling variants of this product

BrdU assay (22 citations) BrdU incorporation (2 citations) Bromodeoxyuridine (BrdU) Incorporation Assay Kit (2 citations) Brdu) incorporation assay kits (2 citations) ELISA BrdU incorporation assay (2 citations)

12 protocols using brdu incorporation assay

1

Colorimetric BrdU Assay for DNA Synthesis

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DNA synthesis was assessed using a colorimetric BrdU incorporation assay (Sigma-Aldrich, Taufkirchen, Germany). Briefly, HaCaT keratinocytes were seeded on 96-well-plates in quintuplicate (2×104 per well) and starved overnight. Approximately 12 h later, the cells were treated with the respective test substances and BrdU labeling solution for 4 h. Subsequently, the assay was carried out according to the manufacturers protocol. Finally, after 15 min incubation with substrate solution, absorbance was measured at 370 nm (reference wavelength 492 nm) using the Infinite 200 PRO plate-reader (Tecan, Maennedorf, Switzerland).
Vogeley C., Sondermann N.C., Woeste S., Momin A.A., Gilardino V., Hartung F., Heinen M., Maaß S.K., Mescher M., Pollet M., Rolfes K.M., Vogel C.F., Rossi A., Lang D., Arold S.T., Nakamura M, & Haarmann-Stemmann T. (2021). Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response. Environment international, 158, 106989.
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2

Colorimetric BrdU Assay for DNA Synthesis

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DNA synthesis was assessed using a colorimetric BrdU incorporation assay (Sigma-Aldrich, Taufkirchen, Germany). Briefly, HaCaT keratinocytes were seeded on 96-well-plates in quintuplicate (2×104 per well) and starved overnight. Approximately 12 h later, the cells were treated with the respective test substances and BrdU labeling solution for 4 h. Subsequently, the assay was carried out according to the manufacturers protocol. Finally, after 15 min incubation with substrate solution, absorbance was measured at 370 nm (reference wavelength 492 nm) using the Infinite 200 PRO plate-reader (Tecan, Maennedorf, Switzerland).
Vogeley C., Sondermann N.C., Woeste S., Momin A.A., Gilardino V., Hartung F., Heinen M., Maaß S.K., Mescher M., Pollet M., Rolfes K.M., Vogel C.F., Rossi A., Lang D., Arold S.T., Nakamura M, & Haarmann-Stemmann T. (2021). Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response. Environment international, 158, 106989.
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3

Evaluating Cell Proliferation Post-Radiation

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BrdU incorporation assay (Sigma-Aldrich, Saint Louis, Missouri, USA) was used to assess cell proliferation based on DNA synthesis 96 h after exposure to radiating microspheres in normoxia according to the manufacturer’s protocol. Absorbance was measured using a plate spectrophotometer (MultscanGO, ThermoFisher Scientific, Waltham, MA, USA) at 450 nm. Results were compared to those of control cells normalized to 100%.
Piasecki P., Majewska A., Narloch J., Maciak M., Brodaczewska K., Kuc M., Was H., Wierzbicki M., Brzozowski K., Ziecina P., Mazurek A., Dziuk M., Iller E, & Kieda C. (2021). A new in vitro model applied 90Y microspheres to study the effects of low dose beta radiation on colorectal cancer cell line in various oxygenation conditions. Scientific Reports, 11, 4472.
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4

Treg Suppression of T Cell Proliferation

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CD4+CD25+ Tregs (5 × 104) from IL-2 treated and saline injected BALB/c hosts and CD4+CD25− responder cells (1 × 105) from naïve BALB/c mice were isolated from the DLNs using magnetic separation, then co-cultured for 5 days in the presence of 1 × 105 APCs (isolated by magnetic sorting) from B6 or C3H splenocytes, the latter to provide unrelated (third party) antigen. It was determined through our preliminary studies that Teff to Treg ratio of 2 to 1 would result in 50% reduction in T cell proliferation; therefore, we used same numbers of Teffs and Tregs in our experiments (SDC Figure 2). Proliferation of responder T cells was measured using the BrdU incorporation assay (Millipore, Billerica, MA).
Tahvildari M., Omoto M., Chen Y., Emami-Naeini P., Inomata T., Dohlman T.H., Kaye A.E., Chauhan S.K, & Dana R. (2016). In vivo expansion of regulatory T cells by low-dose interleukin-2 treatment increases allograft survival in corneal transplantation. Transplantation, 100(3), 525-532.
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5

VEGF-A and Cytokine Effects on MS-1 VEC Proliferation

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Proliferation of MS-1 VECs stimulated with 20ng/ml VEGF-A and treated with different doses of recombinant IFNγ (1, 100, 500 ng/ml) (Prepotech, Rock Hill, NJ) or 10ng/mL of recombinant IL-1β (Biolegend) (as a positive control) was assessed using the BrdU incorporation assay (Millipore).
Di Zazzo A., Tahvildari M., Subbarayal B., Yin J., Dohlman T.H., Inomata T., Mashaghi A., Chauhan S.K, & Dana R. (2017). Proangiogenic Function of T Cells in Corneal Transplantation. Transplantation, 101(4), 778-785.
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6

Quantitative Analysis of Cell Proliferation

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A CyQUANT ® cell proliferation assay was used to assess the number of cells present in culture after the different treatments, according to the manufacturer’s protocol. Cells were lysed, and the number of cells was determined from a standard curve of known cell number and intensity, using fluorescence with CyQUANT® GR dye, which strongly binds to nucleic acids.
A BrdU incorporation assay (Millipore, Billerica, MA) was also used to test the effect of GDNF treatment on motor and sensory-derived SC proliferation. SCs were seeded onto PLL-coated 96 well plates at 3,000 cells/well and cultured in SC media with 0, 50, or 100 ng/mL of GDNF for 3 and 7 days. BrdU measurements were performed according to manufacturer’s instructions. Briefly, 24 hours prior to reading, BrdU reagent was added to culture media. Cells were fixed and subsequently stained with BrdU detection antibody and then an IgG- peroxidase conjugate secondary antibody. Absorbance measurements were read using a Multiskan RC plate reader at 450 and 590 nm.
Jesuraj N.J., Marquardt L.M., Kwasa J.A, & Sakiyama-Elbert S.E. (2014). Glial cell line-derived neurotrophic factor promotes increased phenotypic marker expression in femoral sensory and motor-derived Schwann cell cultures. Experimental neurology, 0, 10-18.
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7

Blocking IFNγ and VEGF-A in T Cell-VEC Co-culture

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A neutralizing anti-IFNγ antibody (1mg/mL; LEAF™ purified anti-mouse IFNγ antibody, Biolegend) or an anti-VEGF-A antibody (Biolegend, San Diego, CA) and their respective isotype control antibodies (anti-IFNγ: LEAF™ purified armenian Hamster IgG isotype ctrl antibody, Biolegend; anti-VEGF-A: LEAF™ purified rat IgG2a, κ isotype Ctrl, Biolegend, San Diego, CA) were used for in vitro blockade of IFNγ or VEGF-A, respectively, in our culture system with T cells and VECs. After 24 hours of culture VECs proliferation was assessed using the BrdU incorporation assay (Millipore). After anti-IFNγ treatment we also assessed VEGF-A expression by ELISA.
Di Zazzo A., Tahvildari M., Subbarayal B., Yin J., Dohlman T.H., Inomata T., Mashaghi A., Chauhan S.K, & Dana R. (2017). Proangiogenic Function of T Cells in Corneal Transplantation. Transplantation, 101(4), 778-785.
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8

Comprehensive Cell Viability Analysis

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Cell proliferation was determined by the BrdU incorporation assay (EMD Millipore Corporation, Germany). Cell viability was measured using the Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI). Cell death levels were measured with the Cytotoxicity lactate dehydrogenase Detection Kit (Roche, Mannheim, Germany). A GloMax®-96 Microplate illuminometer (Promega, Madison, WI) was used to read the 96-well plates. Cell apoptosis rates were detected by the TUNEL assay (Abcam, Cambridge, UK).
Dai J., Zhou Q., Chen J., Rexius-Hall M.L., Rehman J, & Zhou G. (2018). Alpha-enolase regulates the malignant phenotype of pulmonary artery smooth muscle cells via the AMPK-Akt pathway. Nature Communications, 9, 3850.
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9

Allogeneic Tregs Suppress Tconv Proliferation

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Conventional T cells (Tconv; 1 × 105) isolated via MACS sort from the dLNs of naïve BALB/c mice were cocultured with Tregs (5 × 104) from transplant recipients (14 days post-transplantation), T cell-depleted allogeneic splenocytes from C57BL/6 or C3H mice (1 × 105), and 1 μg/ml anti-CD3 antibody (145–2C11, BioLegend) for 3 days. Proliferation was measured using the BrdU incorporation assay (EMD Millipore, Billerica, MA, USA), and percent suppression was calculated using the following formula: % suppression = [(Tconv proliferation without Tregs – Tconv proliferation with Tregs)/ (Tconv proliferation without Tregs)] ×100. Percent suppression of Tregs from low-risk recipients cocultured with Tconv and donor APCs was set 100%.
Inomata T., Hua J., Di Zazzo A, & Dana R. (2016). Impaired Function of Peripherally Induced Regulatory T Cells in Hosts at High Risk of Graft Rejection. Scientific Reports, 6, 39924.
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10

Cell Proliferation on Hydrogel Substrates

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ACLFs from different rats (n = 3) were separately cultured on the surface of the SIS hydrogel (n = 3) and the pure collagen hydrogel (n = 3) in 96-well plates at 105 cells/well with Dulbecco's modified Eagle's medium and the different concentrations of FBS described above for 3 days and 7 days. In addition, for the purpose of SEM study, 2 × 106 cells/well were seeded on both hydrogels with 10% FBS in Dulbecco's modified Eagle's medium in six-well plates for 3 days. Cell attachment and morphology were observed with light microscopy and SEM at Day 3. For the cell proliferation assay, a bromodeoxyuridine (BrdU) incorporation assay (EMD Millipore, Billerica, MA, USA) was performed to measure cell proliferation as previously described [28] (link). Briefly, after incubation for 4 hours with BrdU, collagenase was added for hydrogel digestion. Cells were isolated through centrifugation and followed by incubation with anti-BrdU antibodies for BrdU detection. The plate was read on a microplate reader (Infinite M200; Tecan) with excitation at 325 nm and emission at 420 nm.
Liang R., Yang G., Kim K.E., D'Amore A., Pickering A.N., Zhang C, & Woo S.L. (2015). Positive effects of an extracellular matrix hydrogel on rat anterior cruciate ligament fibroblast proliferation and collagen mRNA expression. Journal of Orthopaedic Translation, 3(3), 114-122.
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