HUVECs (100,000 cells) were plated on coverslips in 0.5% FBS EndoGro for 24 h to synchronize cells, followed by incubation with 5 μM Bromodeoxyuridine (BrdU) with or without VEGF (20 ng/mL) for 24 h. The BrdU incorporation were detected using In Situ Cell Proliferation kit, FLUOS (Roche) with minor modification [31 (link)]. The cells were photographed with fluorescence microscope (Keyence, BZ-X700) using × 40 objective. The percentage of BrdU-labelled cells was determined by counting > 400 nuclei per samples as BrdU-positive/total nuclei.
In situ cell proliferation kit
Manufactured by Roche
Sourced in Germany
The In Situ Cell Proliferation kit is a laboratory tool designed to detect and quantify proliferating cells. It provides a method for visualizing cells undergoing DNA synthesis, a key indicator of cell division and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Bz x700, by Keyence (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti cd31, by Abcam (1 mentions)
Anti vwf, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
5 protocols using in situ cell proliferation kit
1
Endothelial Cell Proliferation Assay
2
Evaluating Cell Proliferation in Murine Lungs
3
In Situ Cell Proliferation Assay
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The In Situ Cell Proliferation Kit, FLUOS (Roche, Mannheim, Germany) was used as per manufacturer's instructions. Briefly, animals were injected i.p. with bromo-deoxyuridine (BrdU) labelling reagent 16 h prior to tissue sampling. Immunostaining was also performed as instructed with the addition of 1∶100 rhodamine Griffonia simplicifolia lectin-1 (Vector Laboratories Inc., Burlingame, CA) to allow co-localisation of BrdU-labelled cells with vascular structures. Slides were mounted using 1∶10 Vectorshield plus DAPI: PBS to permit confirmation of nuclear localisation of BrdU-labelling.
4
Mouse Lung Cell Proliferation Assay
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BrdU (75 mg/kg BW, Sigma-Aldrich) was intraperitoneally injected to mice 4 h prior to tissue collection. Mouse lung cryosections (6 µm thick) were stained with FITC-conjugated anti-BrdU antibody using the In Situ Cell Proliferation kit (Roche Diagnostics) [31] (link) and nuclei were counterstained with DAPI. Anti-vWF (1∶300, Sigma) and anti-CD31 antibodies (1∶40, Abcam, Cambridge, MA) were used to identify endothelial cells.
5
In vivo BrdU Labeling and Imaging
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For in vivo labeling with (bromodeoxyuridine) BrdU, 10 µl of the labeling reagent per 1 g body weight was injected into the hemocoel of staged larvae/pupae. Midguts were dissected 4 h later and then fixed in 10% neutral buffered formalin for 8 h. Paraffin sections of midgut samples were prepared as described above for histological analysis. The sections were processed using In Situ Cell Proliferation Kit, Fluos (Roche, Penzberg, Germany) according to manufacturer's instructions. The percentage of BrdU positive cells was assessed by Image J analysis (National Institutes of Health, Bethesda, MD, USA). Automated particle counting analyze was performed for all images and percentage of positive cells were calculated according to obtained results.
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