The hCMEC/D3 or M059K cells were seeded at 1 × 104 cells in 100 μl of medium per well into 96-well plates and serum-starved for 24 h, incubating with 100 ng/ml γhNRG-1 for 30 min, followed by exposing to heme at 30 μM or 0.02 μg/ml CXCL10 for 24 h. MTT assay was performed in accordance to the manufacturer’s instructions. Ten microliters of MTT reagent (the ratio of MTT reagent to medium is 1:10) was added into each well and incubated in the dark at room temperature for 2 to 4 h. Absorbance at 570 nm was measured using 650 nm as reference filter by a CytoFluorTM 2300 plate reader and the software CytoFluorTM 2300 v. 3A1 (Millipore Co, Bedford, MA, USA).
Cytofluor 2300 v 3a1
Manufactured by Merck Group
Sourced in United States
The CytoFluorTM 2300 v. 3A1 is a multi-functional fluorescence-based microplate reader. It is designed to measure fluorescence intensity, luminescence, and absorbance in a variety of plate formats. The device features advanced optics and a high-performance photomultiplier tube to provide sensitive and accurate measurements.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using cytofluor 2300 v 3a1
1
Cell Viability Assay with Growth Factors
2
Heme Cytotoxicity Evaluation in BeWo Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BeWo cells were seeded at 1 × 104 cells in 100 μl of medium per well into 96-well plates and serum-starved for 24 h, followed by exposing to heme at 10, 20, 30, 60 and 90 μM for 24 h. MTT assay was performed in accordance to the manufacturer’s instructions. 10 μl of MTT reagent (the ratio of MTT reagent to medium is 1:10) was added into each well and incubated in the dark at room temperature for 2 to 4 h. Absorbance at 570 nm was measured using 650 nm as reference filter by a CytoFluorTM 2300 plate reader and the software CytoFluorTM 2300 v. 3A1 (Millipore Co, Bedford, MA, USA).
3
Cell Proliferation Assay with CCK-8
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Cell counting kit (CCK-8) obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD), is a convenient and sensitive method for cell proliferation assays. It utilizes a highly water-soluble tetrazolium salt, WST-8, which produces a water-soluble formazan dye upon reduction in the presence of an electron mediator. The amount of the formazan generated from WST-8 reduction by dehydrogenases is directly in proportion to the numbers of living cells. After seeding 10,000 cells/well in Geltrex-coated 96 wells plates, they were allowed to adhere and proliferate for 48 hours until reaching 80% confluence, then HRP2, anti-HRP2/ anti-TLR1/anti-TLR2 antibodies (Fisher Scientific) or NRG1 were added. The already mixed CCK-8 solution was subsequently added to the wells, the plates were placed in the incubator for 30 min-1 hour, then absorbance at 450 nm was read using a microplate reader [CytoFluorTM 2300 plate reader and CytoFluorTM 2300 v. 3A1 software (Millipore Co, Bedford, MA]). The absorbance was plotted to determine cell proliferation.
4
Assessing iPSC Viability with Heme and NRG-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viability of iPSCs treated with heme and with or without NRG-1 was assessed by determining the number of live cells by colorimetry. The cell counting kit-8 obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD) uses highly water-soluble tetrazolium salt, WST-8, which is reduced by dehydrogenase activity in cells to produce yellow formazan dye, which is soluble in tissue culture medium. The amount of formazan dye is directly proportional to the number of living cells. iPSCs (10,000 cells/well) were treated with heme (0, 10, 20, 30, 60, 90 μM) for 8 hours, then NRG-1 (100 ng/ml) for 18 hours, and then 10 μl of CCK-8 solution was added for 1 hour. Camptothecin (CPT) was used as a positive control for cell toxicity. The optical density of the colorimetric reaction was determined at 450 nm by a CytoFluorTM 2300 plate reader and CytoFluorTM 2300 v. 3A1 software (Millipore Co, Bedford, MA). The absorbance was plotted versus heme and NRG-1 treatment to determine cell viability.
5
Longitudinal Apoptosis and Necrosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess early apoptosis/necrosis longitudinally in iPSC cultured with HRP2/anti-TLR1 or anti-TLR2 antibodies, we used the RealTime-Glo Annexin V Apoptosis and Necrosis Assay obtained from Promega (Madison, WI) following the manufacturer’s instructions. Briefly, iPSC’s were placed in 96-well low attachment plates, and then Annexin NanoBit substrate, CaCl2, necrosis detection reagent, Annexin V-SmBiT and AnnexinV-LgBiT were added. HRP2-induced apoptosis was measured every hour for 6 hours, then at 24 and 26 hours by using a plate reader [CytoFluorTM 2300 plate reader and CytoFluor 2300 v. 3A1 software (Millipore)] set for chemiluminescence, while necrosis was detected by setting up the plate reader for green fluorescence (excitation 485 nm, emission 525 nm).
6
Apoptosis and Necrosis Assessment in Cortical Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess early apoptosis/necrosis in cortical organoids, they were cultured for 20 and 40 days in maturation medium, and then treated with heme for 8 hours followed by NRG-1 for 18 hours. CPT was used as a positive control for apoptosis. We used the RealTime-Glo Annexin V Apoptosis and Necrosis Assay obtained from Promega (Madison, WI) following the manufacturer’s instructions. Briefly, the organoids were placed in 96-well low attachment plates, and then Annexin NanoBit substrate, CaCl2, necrosis detection reagent, Annexin V-SmBiT and AnnexinV-LgBiT were added. Heme-induced apoptosis was measured by using a plate reader [CytoFluorTM 2300 plate reader and CytoFluor 2300 v. 3A1 software (Millipore)] set for chemiluminescence, while heme-induced necrosis was detected by setting up the plate reader for green fluorescence (excitation 485 nm, emission 525 nm). The readings were normalized to the organoid volume, as measured by the formula (Lxw2)/2, where L is the large diameter and w is the small diameter of each organoid. L and were measured using ImageJ software. Green fluorescence images of heme-induced necrosis in cortical organoids were collected by using a Zeiss 700 confocal laser scanning microscope (LSM) (Carl Zeiss, Wetzlar, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!