Human aortic endothelial cells (HAECs) were acquired from Lifeline Cell Technology (Frederick MD). HAECs were cultured in VascuLife EnGS Complete Medium (Lifeline Cell Technology) prior to detachment with 0.05% trypsin-EDTA and plating in a 96 well plate at 10,000 cells/well. Following overnight cell attachment, the growth medium was replaced with EnGS complete medium with 1% penicillin-streptomycin-amphotericin (PSA, LS-1085, Lifeline Cell Technology) containing 1 U/ml human thrombin (Haematologic Technologies, Essex Junction, VT) plus equimolar amounts of free PPACK, precursor NP and PPACK-NP. The cells were allowed to incubate for 4 days with gentle shaking to prevent precipitation of nanoparticles. Following 4 days of incubation, proliferation of HAECs was determined using an XTT assay (Biotium Inc., Hayward, CA) as per the manufacturers instructions.
Xtt assay
Manufactured by Biotium
Sourced in United States
The XTT assay is a colorimetric method used to measure cell viability and proliferation. It utilizes the tetrazolium salt XTT, which can be reduced by metabolically active cells to form an orange formazan product that can be quantified spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Human thrombin, by Prolytix (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
Victor3, by PerkinElmer (1 mentions)
Trametinib, by Merck Group (1 mentions)
Dabrafenib, by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Celltiter glo, by Promega (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Human il 4, by Thermo Fisher Scientific (1 mentions)
7 protocols using xtt assay
1
Thrombin-Induced Proliferation of Aortic Endothelial Cells
2
Cytotoxicity Evaluation via XTT Assay
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Cytotoxicity was analyzed by XTT assay (Biotium, Fremont, CA, USA). In short, RAW 264.7 cells were seeded in 96-well plates and treated with antioxidants or pro-oxidant. RAW 264.7 cells were treated with RES (TCI, Tokyo, Japan) and MT (Sigma-Aldrich, Darmstadt, Germany) for 4 days, whereas the cells were treated with DOX (TCI) for 3 h. Hereafter, 25 µL of XTT activator reagent was mixed with 5 mL XTT reagent and mixed. Next, 50 µL of freshly prepared XTT reagent (activator + reagent) was added to 100 µL of fresh media 100. Finally, 150 µL of media + XTT was added to each well and incubated for 2–4 h at 37 °C, 5% CO2. The absorbance was measured at 630 nm using a microplate reader (Biotek synergy 4, Winooski, VT, USA). Three isolated experiments were performed.
3
Drug Synergy Evaluation in Cancer Cells
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MBZ, dabrafenib, and trametinib were purchased from Sigma-Aldrich and ActiveBiochem, respectively. 5 × 103 viable cells per well were plated in 96-well dishes and allowed to recover for 12 h prior to drug treatment. Cells in triplicate wells were treated for up to 72 h (based on initial time course experiments showing maximal effects at that time point) with different concentrations of trametinib, dabrafenib, or MBZ alone, or a combination of MBZ and trametinib. Negative controls were exposed to vehicle DMSO in the same volumes. Cell viability was assessed by an XTT assay, according to a manufacturer's specifications (Biotium Inc). Reduced XTT was measured by absorbance at 490 nm on a PerkinElmer Victor3 plate reader. Cells exposed to detergent served as a positive control.
4
Metabolic Activity Evaluation of Cell-Laden Microspheres
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The metabolic activity
of cell-laden microspheres was assessed using the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide
(XTT) assay (Biotium) according to the manufacturer’s protocol.
Cell-laden microspheres were transferred into a 96-well-plate with
one microsphere per well in 100 μL of media. 25 μL of
the XTT working solution was added to each well. After incubation
for 18 h at 37 °C, absorbance (470 nm) and background (660 nm)
intensities were measured via a microplate reader (Bio-Tek). The background
intensity was subtracted from the absorbance intensity to obtain the
relative absorbance intensity for each microsphere. A minimum of five
microspheres were analyzed per condition at each time point. The relative
metabolic rate obtained was normalized to day 0 values for each cell
type.
of cell-laden microspheres was assessed using the 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide
(XTT) assay (Biotium) according to the manufacturer’s protocol.
Cell-laden microspheres were transferred into a 96-well-plate with
one microsphere per well in 100 μL of media. 25 μL of
the XTT working solution was added to each well. After incubation
for 18 h at 37 °C, absorbance (470 nm) and background (660 nm)
intensities were measured via a microplate reader (Bio-Tek). The background
intensity was subtracted from the absorbance intensity to obtain the
relative absorbance intensity for each microsphere. A minimum of five
microspheres were analyzed per condition at each time point. The relative
metabolic rate obtained was normalized to day 0 values for each cell
type.
5
Antibody-drug conjugate cytotoxicity assay
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The SEMA4A antibody (5E3) was directly conjugated to either monomethyl auristatin E using a mc-vcPAB linker (maleimide-based linker, cysteine linked) or with mertansine (DM1) using an SMCC linker (N-hydroxysuccinimide–ester based, lysine linked) by Abzena. An isotype-matched control, trastuzumab, directly conjugated to monomethyl auristatin E (mc-vcPAB linker) was also provided by Abzena.
Cells were seeded at 5 × 103 per well in a 96-well plate in complete RPMI 1640. Antibody-ZAP complexes (Advanced Targeting Systems; produced according to manufacturer's instructions) or ADCs were added to the cells and plates incubated for 72 hours at 37°C and 5% carbon dioxide. Cell viability was determined by XTT assay (Biotium) as per the manufacturer's guidelines and calculated as a percentage of the control (media only) wells. For primary cells, CD138+ and CD14+ cells were isolated by using microbeads (Miltenyi Biotec), seeded at 5 to 10 × 103 per well and incubated for 72 hours with ADC or vehicle. Cell viability was determined by using CellTiter-Glo (Promega) per the manufacturer's guidelines or by flow cytometry using Annexin V and LIVE/DEAD (Thermo Fisher Scientific) staining.
Cells were seeded at 5 × 103 per well in a 96-well plate in complete RPMI 1640. Antibody-ZAP complexes (Advanced Targeting Systems; produced according to manufacturer's instructions) or ADCs were added to the cells and plates incubated for 72 hours at 37°C and 5% carbon dioxide. Cell viability was determined by XTT assay (Biotium) as per the manufacturer's guidelines and calculated as a percentage of the control (media only) wells. For primary cells, CD138+ and CD14+ cells were isolated by using microbeads (Miltenyi Biotec), seeded at 5 to 10 × 103 per well and incubated for 72 hours with ADC or vehicle. Cell viability was determined by using CellTiter-Glo (Promega) per the manufacturer's guidelines or by flow cytometry using Annexin V and LIVE/DEAD (Thermo Fisher Scientific) staining.
6
Endothelial and T-ALL Cell Viability Assay
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For endothelial cells, 96-well plates were seeded with 4 × 103 HUVEC or mLMVEC cells (Lonza), with indicated concentrations of either human IgG Fc (Sino Biologics), N110-14Fc, or N410-14Fc. Each concentration is represented by six replicates. For T-ALL cells, KopTK or T6E T-ALL cells were seeded at 8 × 103 cells per well in a 96-well plate in RPMI media and treated with the indicated concentration of IgG Fc, N110–14Fc, or N410–14Fc. After incubation for 72 h, cell viability was determined by XTT assay (Biotium).
7
Quantifying Recombinant Human IL-10 Activity
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Example 12
The purified recombinant human IL-10 was quantified using a commercial ELISA for human IL-10 (Biolegend). The biological activity of the expressed IL-10 was tested by using a dose-dependent co-stimulation (with human IL-4) of MC/9 cell proliferation. Briefly, cells were spun down and washed twice with RPMI1640 (Gibco) to remove all traces of the Rat-T-STIM in the growth medium. Next, cells were re-suspended in DMEM supplemented with 10% FBS, 2 mM glutamate, pen/strep, 2-meracaptoethanal and 200 pg/ml human IL-4 (Peprotech) and were plated at a density of 20,000 cells per well in a 96-well plate. A commercial recombinant human IL-10 (Peprotech) was used to generate a standard curve. Cells were grown for 82 hours and cell proliferation was determined using an XTT assay (Biotium).
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