In vitro antitrypanosomal activities against T. b. gambiense strain was described by Otoguro et al [25 (link)]. In brief, T. b. gambiense strain was cultured in IMDM with various supplements and 10% heat-inactivated FBS at 37°C, under 5.0% CO2/95% air according to the method of Baltz et al [26 ]. Ninety five mL of the trypanosomes suspension (2.0–2.5104 trypanosomes/mL for strain GUTat 3.1) was seeded in a 96-well microplate, and 5.0 mL of a test compound solution (dissolved in 5.0% dimethylsulfoxide) was added followed by incubation for 72 h (long incubation-low inoculation test: LILIT). Ten mL of the fluorescent dye Alamar Blue was added to each well. After incubation for 3–6 h, the resulting solution was read at 528/20 nm excitation wavelength and 590/35 nm emission wavelength by a FLx800 fluorescent plate reader (Bio-Tek Instrument, Inc. Vermont, USA). Data were transferred into a spreadsheet program (Excel). The IC50 values were determined using fluorescent plate reader software (KC-4, Bio-Tek). Successive subcultures were done in 24-well tissue culture plates under the same conditions.
Flx800 fluorescent plate reader
Manufactured by Agilent Technologies
Sourced in United States
The FLx800 is a fluorescent plate reader designed to measure fluorescence in microplates. It is a versatile instrument capable of detecting a wide range of fluorescent signals.
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Lab products found in correlation
Caspase 3 activity assay kit, by Cell Signaling Technology (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
96 well transwell migration assay, by Corning (2 mentions)
Sdf 1α, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Pathscan sandwich elisa lysis buffer, by Cell Signaling Technology (1 mentions)
Transwell migration assay, by Corning (1 mentions)
8 protocols using flx800 fluorescent plate reader
1
In vitro Antitrypanosomal Activity Assay
2
Microglial Phagocytosis of Aβ42
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Microglia were immediately used upon isolation by stimulating in serum-free DMEM/F12 with or without 100 ng/ml IGF-1, 1 μM FITC-human Aβ42, or FITC-Escherichia coli bioparticle as a positive control, 0.125 mg/ml. Phagocytosis was measured by the uptake of fluorescein isothiocyanate (FITC)-conjugated Aβ42. The Aβ42 peptide was fibrillized as recommended by the manufacturer (rPeptide, Athens, GA, USA) and previously described (Floden and Combs, 2006 (link)). Cells were treated for 6 h, media was removed, and the wells were rinsed with 0.25 mg/ml trypan blue in PBS to quench any extracellular peptide or bioparticles that were not internalized. The intracellular fluorescence was read at 480 nm excitation and 520 nm emission via Bio-Tek FLx800 fluorescent plate reader (Winooski, VT, USA).
3
Caspase-3 Activity Assay Protocol
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The caspase-3-like activity was determined using the Caspase-3 Activity Assay kit (Cell Signaling Technology) according to the manufacturer’s protocol. Briefly, 50 μL of lysates were added to 150 μL of assay buffer containing a fluorogenic substrate [1 mg/mL N-acetyl-Asp-Glu-Val-Asp-(7-amino-4-methylcoumarin)(Ac-DEVD-AMC)], which was cleaved by caspase-3. Accumulation of AMC fluorescence was monitored over 180 min using an FLx800 fluorescent plate reader (BioTek, Winooski, VT, USA) at excitation wavelength 380 nm and emission wavelength 460 nm. Fluorescence of each sample reading at time 0 h was subtracted from the detected value. AMC fluorescence (relative fluorescence units, RFU) of each sample was normalized to the protein content of the extracts. Caspase-3-like activity from the untreated sample was arbitrarily set at 1 for the calculation of fold induction.
4
Cytotoxicity Assay for MRC5 and T. b. brucei
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Measurement
of inhibition of the proliferation of MRC5 (human lung fibroblast)
cells and T. b. brucei bloodstream
stage cells was performed using a modification of the cell viability
assay previously described.33 (link) Compounds
(50 μM to 0.5 nM) were incubated with 2 × 103 cells/well in 0.2 mL of the appropriate culture medium (MEM with
10% fetal bovine serum for MRC5 cells) in clear 96-well plates. Plates
were incubated at 37 °C in the presence of 5% CO2 for
69 h. Resazurin was then added to a final concentration of 50 μM,
and plates were incubated as above for a further 4 h before being
read on a BioTek flx800 fluorescent plate reader.
of inhibition of the proliferation of MRC5 (human lung fibroblast)
cells and T. b. brucei bloodstream
stage cells was performed using a modification of the cell viability
assay previously described.33 (link) Compounds
(50 μM to 0.5 nM) were incubated with 2 × 103 cells/well in 0.2 mL of the appropriate culture medium (MEM with
10% fetal bovine serum for MRC5 cells) in clear 96-well plates. Plates
were incubated at 37 °C in the presence of 5% CO2 for
69 h. Resazurin was then added to a final concentration of 50 μM,
and plates were incubated as above for a further 4 h before being
read on a BioTek flx800 fluorescent plate reader.
5
CPC Migration Assay Protocol
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CPCs were trypsinized, counted, and resuspended in starving medium that contained IMDM with GlutaMAX (ThermoFisher Scientific, Waltham, MA), 1% Insulin–Transferrin–Selenium (Life Technologies, Carlsbad, CA), and 0.5% FBS (Thermo Scientific, Waltham, MA). 50,000 CPCs were plated in the top chamber of a 96-well transwell migration assay (Corning, Union City, CA) with 8 μm pores. Standard CPC growth medium supplemented with 100 ng/mL of SDF-1α (Life Technologies, Grand Island, NY) was used in the bottom chamber as a chemoattractant. After 6 h, migrated cells in the bottom chamber were stained using Calcein AM (Fisher Scientific Pittsburg, PA) and quantified using a FLX800 fluorescent plate reader (Bio-Tek, Winooski, VT).
6
Caspase-3 Activity Assay in SH-SY5Y Cells
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SH-SY5Y cells were harvested in caspase lysis buffer (PathScan®®®® sandwich ELISA lysis buffer, Cell Signaling Technology). The caspase-3-like activity was assayed using the Caspase-3 Activity Assay kit (Cell Signaling Technology) according to the manufacturer’s instructions. Briefly, 50 μL of lysates were added to 150 μL of assay buffer containing 1 mg/mL N-acetyl-Asp-Glu-Val-Asp-(7-amino-4-methylcoumarin) (Ac-DEVD-AMC), a fluorogenic substrate cleaved by caspase-3. Accumulation of AMC fluorescence was monitored over 180 min using an FLx800 fluorescent plate reader (BioTek, Winooski, VT, USA) at excitation and emission wavelengths of 380 and 460 nm, respectively. Fluorescence of each sample reading at time 0 h was subtracted from the determined value. AMC fluorescence (Relative fluorescent units, RFU) of each sample was normalized to the protein content of the extracts. Caspase-3-like activity from the untreated sample was arbitrarily set at 1 for the calculation of fold induction.
7
Cytotoxicity Assay for Trypanosoma and Human Cells
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Measurement of the ability of
the compounds to inhibit human (MRC5, human lung fibroblast cells)
and trypanosome (T. b. brucei, BSF427,
VSG118) cell growth was performed using a modification of the cell
viability assay previously described by Raz et al.29 (link) Compounds were dissolved in DMSO at a top concentration
of 10 mM and serially diluted in half log steps to achieve a range
of final assay concentrations of 50 μM to 0.5 nM. Compound at
each concentration (200-fold final) was added to clear 96-well tissue
culture plates in a volume of 1 μL. Then 2000 cells per well
in relevant growth medium (HMI-9T for T. brucei, a modification of HMI-9 as described by Hurumi et al.,30 (link) where 0.2 mM 2-mercaptoethanol was replaced
with 0.056 mM thiolglycerol, and MEM with 10% FBS for MRC-5) were
then added to columns 1–11 of the plates in a volume of 199
μL. To column 12, 200 μL of medium was added to provide
a no cells control. Plates were then incubated at 37 °C in an
atmosphere of 5% CO2 for 69 h before the addition of 20
μL of 500 μM rezasurin solution and a further incubation
period of 4 h. Plates were then read on a BioTek flx800 fluorescent
plate reader, and percentage inhibition was compared to the maximum
and minimum assay controls. Concentration effect curves were fitted
using nonlinear regression using XLFit 4.2 and EC50 values
determined.
the compounds to inhibit human (MRC5, human lung fibroblast cells)
and trypanosome (T. b. brucei, BSF427,
VSG118) cell growth was performed using a modification of the cell
viability assay previously described by Raz et al.29 (link) Compounds were dissolved in DMSO at a top concentration
of 10 mM and serially diluted in half log steps to achieve a range
of final assay concentrations of 50 μM to 0.5 nM. Compound at
each concentration (200-fold final) was added to clear 96-well tissue
culture plates in a volume of 1 μL. Then 2000 cells per well
in relevant growth medium (HMI-9T for T. brucei, a modification of HMI-9 as described by Hurumi et al.,30 (link) where 0.2 mM 2-mercaptoethanol was replaced
with 0.056 mM thiolglycerol, and MEM with 10% FBS for MRC-5) were
then added to columns 1–11 of the plates in a volume of 199
μL. To column 12, 200 μL of medium was added to provide
a no cells control. Plates were then incubated at 37 °C in an
atmosphere of 5% CO2 for 69 h before the addition of 20
μL of 500 μM rezasurin solution and a further incubation
period of 4 h. Plates were then read on a BioTek flx800 fluorescent
plate reader, and percentage inhibition was compared to the maximum
and minimum assay controls. Concentration effect curves were fitted
using nonlinear regression using XLFit 4.2 and EC50 values
determined.
8
Clinorotation-Induced Cell Migration Assay
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After exposure to 6–7 days of clinorotation, cells were trypsinized counted, resuspended in starving medium, and plated in the top chamber of a 96-well transwell migration assay (Corning, Union City, CA) with 8 micron pores. The transwell migration assay was performed according to manufacturer’s instructions (Corning, Union City, CA). In brief, cells were plated at a density of 50,000 cells per well in the top transwell chamber and growth medium supplemented with 100ng/mL of SDF-1α was used in the bottom chamber as a chemoattractant (Life Technologies, Grand Island, NY). After 6 hours, migrated cells in the bottom chamber were stained using Calcein AM (Fisher Scientific Pittsburg, PA) and quantified using a FLX800 fluorescent plate reader (Bio-Tek, Winooski, VT).
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