105 HUVECs per well were seeded in an MW24 plate the afternoon before the experiment, and cells were incubated overnight. HUVECs were then pre-treated with different doses of HT-C6 in EGM-2 media 30 min before induction with TNF-α, and cells were incubated for additional 6 h. Moreover, 1 h before the end of the treatments, THP-1 cells were stained with 5 μg/mL calcein (Calbiochem, Merck, Darmstadt, Germany) for 30 min, and were then washed with PBS, centrifuged at 1500 rpm for 5 min, and resuspended in EGM-2 medium. After a wash, HUVECs were co-cultured with the stained THP-1 for 1 h to allow monocyte adhesion. Subsequently, the co-culture was gently washed once with EBM-2 media to remove the non-adhered THP-1, and fresh EGM-2 media was added. Then, fluorescence was measured in an FL600 fluorescence plate reader (BioTek, Charlotte, VT, USA), and bright field and fluorescence images were taken in a fluorescence Nikon Eclipse Ti microscope (Nikon, Minato, Tokio, Japan).
Fl600 fluorescence plate reader
Manufactured by Agilent Technologies
Sourced in United States
The FL600 fluorescence plate reader is a laboratory instrument designed to measure the fluorescence of samples in a microplate format. It provides accurate and reliable fluorescence measurements for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Apo one homogeneous caspase 3 7 assay, by Promega (2 mentions)
Celltiter blue cell viability assay, by Promega (2 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Quant it picogreen assay, by Thermo Fisher Scientific (1 mentions)
Ebm 2 media, by Lonza (1 mentions)
Fluoroblok insert, by Corning (1 mentions)
Caspase fluorometric assay kit, by Abcam (1 mentions)
Celltiter blue cell viability assay g8080, by Promega (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Promega (1 mentions)
10 protocols using fl600 fluorescence plate reader
1
Monocyte Adhesion Assay with HUVECs
2
Caspase-3/7 Activity Quantification
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Caspase-3/7 activity was evaluated immediately after the cell viability determination in the same wells using a synthetic rhodamine-labeled caspase-3/7 substrate (Apo-ONE® Homogeneous Caspase-3/7 Assay, G7790; Promega Corporation). After 60 minutes of cultivation at the room temperature, the fluorescence was measured (ex: 499 nm/em: 512 nm) with an FL600 fluorescence plate reader (Bio-Tek). Caspase-3/7 activity was calculated as the fluorescence readings of the treated group/mock control ×100.33 (link)
3
Quantification of dsDNA Release
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Release of dsDNA in cell-free supernatants was analyzed using the Quant-iTPicogreen assay (Invitrogen, Breda, The Netherlands) and a FL600 fluorescence plate reader (Bio-Tek Instruments) with a wavelength of 485 nm through a 590 nm band pass filter.
4
Cell Viability Assay Using Resorufin
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To confirm the results from the MTS assay, fluorimetric detection of resorufin (CellTiter-Blue Cell Viability Assay, G8080; Promega) was utilized to measure the cell viability. The procedure given in the manufacturer instructions was followed. An FL600 fluorescence plate reader (BioTek, Winooski, VT, USA) was used for fluorimetry analysis (excitation 560 nm/emission 590 nm). Fluorescence data were presented as the fluorescence of treated sample/mock control ×100%.
5
Endothelial Cell Monocyte Migration Assay
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HUVECs were grown to subconfluency in MW6 plates and were then pre-treated for 30 min with different doses of HT-C6 in EBM-2 media (Lonza, Basel, Switzerland) supplemented with 0.2% bovine serum albumin (BSA). After that, induction with 20 ng/mL TNF-α was conducted. Conditioned media were collected after 16 h (overnight incubation), and centrifuged at 1500 rpm for 5 min to remove cellular debris. Concurrently, THP-1 cells were stained with 5 μg/mL calcein (Calbiochem, Merck, Darmstadt, Germany) for 30 min, and were then washed with PBS, centrifuged at 1500 rpm for 5 min and resuspended in EBM-2 medium. Next, 700 μL of conditioned media were placed on wells, onto which 8 μM FluoroBlok™️ Inserts (Corning, MA, USA) were placed. Finally, 200 μL of a THP-1 cell suspension (5 × 104 cells) were seeded onto the inserts. The plaque was then incubated for 2 h, and fluorescence was measured in an FL600 fluorescence plate reader (BioTek, Charlotte, VT, USA) every 30 min. The signal was measured in arbitrary units (RFUs). In addition to negative and positive controls of TNF-α induction, negative controls of staining and chemoattraction were included in this experiment.
6
Fluorometric Caspase Activity Assay
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The Caspase 3, 8 and 9 activity assays were performed using Caspase Fluorometric Assay kit (BioVision, USA). The assay is based on estimation of the intensity of fluorescence produced by cleavage of specific sequence (substrate) coupled with a fluorogenic compound, 7-amino-4-trifluoromethyl coumarin (AFC) which serves as a substrate. The tissue homogenate equivalent to 100 µg of total protein was diluted with 2× reaction buffer containing 10 mM DTT. Five microlitre of the respective substrate (Caspase-3: DEVD-AFC; Caspase-8: IETD-AFC; Caspase-9: LEHD-AFC) was added to the reaction mixture in micro-plate and incubated at 37°C for 1–2 h. The fluorescence intensity was measured using Bio-Tek FL600 fluorescence plate reader (Bio-Tek Instruments, Winooski, VT).
7
Caspase-3/7 Activity Assay
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After the measurement of cell viability, caspase-3/7 activity was tested instantly using a synthetic rhodamine-labeled caspase-3/7 substrate (Apo-ONE® Homogeneous Caspase-3/7 Assay, G7790; Promega) on the same samples according to the manufacturer’s instructions. After incubation for 1 hour at room temperature, an FL600 fluorescence plate reader (BioTek) was used to measure the fluorescence of each well. Caspase-3/7 activity was obtained from the fluorescence of treated sample/mock control.
8
Measurement of Intracellular ROS Levels
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After treatment with various concentrations of IL for 30 min, the SKOV-3 cells were incubated with DCFH-DA (20 μM) for another 30 min. The cells were then observed immediately using an IX51S8F fluorescence microscope. The ROS level was also quantified using an FL-600 fluorescence plate reader (BIO-TEK Instruments, Winooski, USA) as previously described
[22] (link).
[22] (link).
9
Resorufin-Based Cell Viability Assay
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Fluorimetric detection of resorufin (CellTiter-Blue Cell Viability Assay, G8080; Promega Corporation, Fitchburg, WI, USA) was used to determine cell viability, as described previously.29 (link) A FL600 fluorescence plate reader (Bio-Tek, Winooski, VT, USA) was used for fluorimetry (ex: 560 nm/em: 590 nm).
10
Evaluating Cell Proliferation via MTT and CellTiter-Blue Assays
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In our current study, cell proliferation was evaluated through an MTT assay. Briefly, SMMC-7721 cells were washed twice with PBS when the cell , density reached 80%. The cell suspension was fabricated after being digested by trypsin, and then, cells were seeded into 96-well plates with 200 μL of volume per well, each of which containing 3 × 10 3 -6 × 10 3 cells. Subsequently, MTT (20 μL, 5 mg/mL; Sigma, USA) was seeded into each well, and the cells were then incubated at 37°C with 5% CO 2 for 4 h. Dimethyl sulfoxide (DMSO; 150 μL) was placed into each well, and the cells were softly shaken for nearly 10 min to dissolve the crystal. A microplate reader was applied to distinguish the absorbance value at 490 nm at 12 h, 24 h, 48 h, 72 h, and 96 h.
Cell viability was evaluated through the fluorimetric detection of resorufin (CellTiter-Blue Cell Viability Assay, Promega, Madison, WI, USA) further confirm the results of the MTT assay. The procedure was performed based on the manufacturer's instructions. An FL600 fluorescence plate reader (Bio-Tek, Winooski, VT, USA) was applied for fluorimetry (excitation = 560 nm, emission = 590 nm). Fluorescence data were calculated as the fluorescence of treated sample/mock control×100%.
Cell viability was evaluated through the fluorimetric detection of resorufin (CellTiter-Blue Cell Viability Assay, Promega, Madison, WI, USA) further confirm the results of the MTT assay. The procedure was performed based on the manufacturer's instructions. An FL600 fluorescence plate reader (Bio-Tek, Winooski, VT, USA) was applied for fluorimetry (excitation = 560 nm, emission = 590 nm). Fluorescence data were calculated as the fluorescence of treated sample/mock control×100%.
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