To study the effect of BET inhibitors on the proliferation of N9 microglial cells, we used a Cell Proliferation BrdU ELISA (colorimetric) Kit (Roche Applied Science, Indianapolis, IN) following manufacturer instructions, as described in our previous study [22 ] with minor modifications. Briefly, N9 cells were seeded in 96-well plates at a density of 4000 cells per well with a final volume of 200 μl, in DMEM containing 0.5% FBS. Cells were pre-treated with 0.5 μM JQ1, 30 μM Olinone, 30 μM RVX208, or an equal volume of vehicle control (DMSO) for 12 h prior to LPS stimulation (final 1 μg/ml). After LPS treatment for 2 h, cells were labeled with BrdU in DMEM containing 10% FBS for a 2-h incubation at 37 °C, and then fixed with a FixDenat solution for 30 min, followed by a 90-min incubation at room temperature with an anti-BrdU-POD antibody (1:100 dilution). After washing with PBS three times, substrate was added. Plates were incubated at room temperature for 30 min, and colorimetric signals were measured on a FlexStation 3 Benchtop Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA) at 370 nm with a reference wavelength of 492 nm.
Cell proliferation brdu elisa colorimetric kit
Manufactured by Roche
Sourced in Germany, Switzerland
The Cell Proliferation BrdU ELISA (colorimetric) Kit is a laboratory tool used to quantify cell proliferation. It measures the incorporation of the nucleoside analog BrdU into the DNA of dividing cells, providing an indirect measure of cell proliferation.
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Lab products found in correlation
Flexstation 3 benchtop multi mode microplate reader, by Molecular Devices (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Ficoll paque, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Propidium iodide, by Beckman Coulter (1 mentions)
Rpmi medium, by Lonza (1 mentions)
Brdu cell proliferation assay kit, by Abcam (1 mentions)
4 protocols using cell proliferation brdu elisa colorimetric kit
1
Microglial Cell Proliferation Assay with BET Inhibitors
2
T-Cell Proliferation Assay with MHC Peptides
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PBMCs from healthy donors and NPC patients were seeded in a 96-well flat bottom culture plate at the density of 2 x 105 cells/ well. Fifty μg/ml of MHC class II peptides (Pep-07 and Pep-08) or 0.5μg/ml of the phytohaemagglutinin (PHA) were added to respective wells and incubated for 3–5 days at 37°C. The proliferation of T-cells was examined using Cell Proliferation BrdU ELISA Colorimetric Kit (Roche Diagnostics GmbH, Germany) as per the manufacturer’s instructions. PBMCs stimulated by PHA were used as a positive control while the wells seeded with PBMCs cultured in the absence of peptide served as negative controls. T-cell proliferation induced by MHC class II peptides was determined by measuring the absorbance at 450 nm after subtracting the negative control wells.
3
Immunomodulatory Potential of ASCs on PBMCs
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The immunomodulatory potential of ASCs on activated peripheral blood mononuclear cells (PBMCs) was studied using a co-culture system of ASCs and PBMCs with a ratio of 10:1 in RPMI medium with 10% FBS (Lonza Group, Rome, Italy). The PBMCs were isolated from healthy donors by density gradient centrifugation with Ficoll-Paque (Sigma-Aldrich, St. Louis, MO, USA) and activated by the addition of phytohemagglutinin (PHA, 1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA). After an incubation of 72 h, the PBMCs were fixed with 70% ethanol and stained with propidium iodide at a concentration of 5 µ/mL (Beckman Coulter, Cassina de’ Pecchi, Milano, Italy) at room temperature for 10 min for cell cycle analysis by flow cytometry. PBMCs without PHA stimulation and PBMCs activated by PHA were analyzed as negative and positive controls, respectively. The immunomodulatory activity of the ASCs was also analyzed by BrdU incorporation in the PBMCs using an ELISA Kit according to the manufacturer instructions (BrdU Cell Proliferation ELISA Colorimetric Kit, Roche, Basel, Switzerland). All experiments were conducted using ASCs derived by NEEGA-DF selection (F-SVFs) directly plated from the SVF (SVF).
4
Assessing Cell Proliferation in HCAECs and MPLECs
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For HCAECs, cells were plated onto a 96-well plate at 3000 cells/well in replicates of 5 and EndMT induced as indicated above. After 24 hours, a BrdU Cell Proliferation ELISA Colorimetric Kit (11647229001, Roche) was used according to the manufacturer’s instructions. BrdU was applied for 10 hours, and absorbance was measured at 370 nm using a SpectraMax M5 microplate reader. The average of 5 experimental replicates was used to calculate the average proliferation rate per HCAEC line.
For MPLECs, cells were treated with 4-OH tamoxifen or vehicle for 2 days and then also with or without EndMT induction for another 2 days (4 treatment conditions in total). Cell were then trypsinized and plated onto a 96-well plate at 3000 cells/well in replicates of 3 and were cultured for a further 2 days with or without induction of EndMT (without 4-OH tamoxifen or vehicle). A BrdU Cell Proliferation Assay Kit (K306-200; BioVision) was then used according to the manufacturer’s instructions. In brief, BrdU was incubated for 4 hours. Absorbance was measured at 650 nm using a SpectraMax M5 microplate reader 25 minutes after TMB substrate was added. The average of 3 experimental replicates was used to measure proliferation for each biological replicate.
For MPLECs, cells were treated with 4-OH tamoxifen or vehicle for 2 days and then also with or without EndMT induction for another 2 days (4 treatment conditions in total). Cell were then trypsinized and plated onto a 96-well plate at 3000 cells/well in replicates of 3 and were cultured for a further 2 days with or without induction of EndMT (without 4-OH tamoxifen or vehicle). A BrdU Cell Proliferation Assay Kit (K306-200; BioVision) was then used according to the manufacturer’s instructions. In brief, BrdU was incubated for 4 hours. Absorbance was measured at 650 nm using a SpectraMax M5 microplate reader 25 minutes after TMB substrate was added. The average of 3 experimental replicates was used to measure proliferation for each biological replicate.
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