Anti-plasmodial activity assays were performed in vitro using 3H-Hypoxanthine (0.5 μCi/well) incorporation assays and P. falciparum 3D7-infected erythrocytes for 48, 72 and 96 h, essentially as previously described [29 (link)]. All assays were carried out in 96-well plates, in triplicate at a single concentration of 4.8 µg/mL and were performed on three separate occasions. Positive antimalarial controls chloroquine (Sigma Aldrich, C6628) and clindamycin (Sigma Aldrich, C5269) were included in each assay. Fast action 48 h assays were performed using synchronous parasites at a 1% parasitemia and 1% haematocrit, whereas 72 and 96 h assays were assessed using synchronous parasites at a 0.25% parasitemia, 2.5% final haematocrit or a 0.1% parasitemia, 2% final haematocrit, respectively. After incubation, assay plates were frozen, thawed and harvested onto 1450 MicroBeta filter mats before counting using a 1450 MicroBeta (PerkinElmer). Growth inhibition was determined by comparing the incorporation of test well to that of untreated vehicle controls (0.5% DMSO).
1450 microbeta
Manufactured by PerkinElmer
Sourced in United States
The 1450 Microbeta is a scintillation counter designed for the detection and quantification of radioactive samples. It utilizes photomultiplier tubes to convert light signals generated by radioactive decay into electrical signals, which are then processed and analyzed. The 1450 Microbeta is capable of measuring various types of radioactive samples, including liquid and solid samples.
Automatically generated - may contain errors
Lab products found in correlation
3h thymidine, by PerkinElmer (3 mentions)
Chloroquine, by Merck Group (1 mentions)
Clindamycin, by Merck Group (1 mentions)
Prism v5, by GraphPad (1 mentions)
Multiscreen fc filter type b, by Merck Group (1 mentions)
Ultima gold, by PerkinElmer (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
Luciferase assay lysis buffer, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Optiphase supermix, by PerkinElmer (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Microbeta 2450, by PerkinElmer (1 mentions)
Facscanto ll, by BD (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Moflo astrios eq flow cytometer, by Beckman Coulter (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Lumaplate 96, by PerkinElmer (1 mentions)
Chromogenic assay, by Siemens (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
18 protocols using 1450 microbeta
1
In Vitro Anti-Plasmodial Activity Assay
2
Treg Cell Specificity Assay Protocol
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The Treg cell specificity assay was performed as described previously [26 (link)], with modifications.
5×103 dendritic cells were pulsed with 200 μg/ml test peptide or human IgG in triplicates. After 14 h of incubation, 2.5×104 CD4+ CD25+ Treg cells were added. Simultaneously, autologous Treg cell-depleted T lymphocytes (Tcon) were polyclonally activated in a plate coated with anti-human CD3 antibody. After 18 h of incubation, 2.5×104 Tcon were added. After 72 h of culture, 3H-thymidine at 37 KBq was added. T cell proliferation was measured by determining the amount of incorporated 3H using the liquid scintillation counter 1450 MicroBeta (PerkinElmer, Waltham Massachusetts, USA).
5×103 dendritic cells were pulsed with 200 μg/ml test peptide or human IgG in triplicates. After 14 h of incubation, 2.5×104 CD4+ CD25+ Treg cells were added. Simultaneously, autologous Treg cell-depleted T lymphocytes (Tcon) were polyclonally activated in a plate coated with anti-human CD3 antibody. After 18 h of incubation, 2.5×104 Tcon were added. After 72 h of culture, 3H-thymidine at 37 KBq was added. T cell proliferation was measured by determining the amount of incorporated 3H using the liquid scintillation counter 1450 MicroBeta (PerkinElmer, Waltham Massachusetts, USA).
3
Peptide Scan Analysis of Antigen-Specific T Cell Responses
4
Proliferation Assay with [3H]Thymidine
5
Quantifying EGFR Binding Affinity
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The affinity
of 64Cu-labeled cetuximab conjugates for EGFR was determined
by a saturation binding assay based on previously published methods.25 (link) HCT116 cell membrane preparations were diluted
in binding buffer [0.1% bovine serum albumin, 50 mM Tris-HCl (pH 7.4),
5.0 mmol/L MgCl2, 0.5 μg/mL aprotinin, 200 μg/mL
bacitracin, 10 μg/mL leupeptin, and 10 μg/mL pepstatin
A], and 15 μg of membrane was added to each well of a 96-well
filtration plate (Multiscreen Durapore; Millipore; Billerica, MA).
Membranes were incubated with increasing concentrations of 64Cu-labeled cetuximab conjugates for 2 h at room temperature. Nonspecific
binding was determined by saturating receptors with excess cetuximab.
When equilibrium was reached, unbound radioactivity was filtered off
and the membranes were washed twice with 200 μL of binding buffer.
Bound radioactivity was measured with a liquid scintillation and luminescence
plate reader (1450 Microbeta; PerkinElmer; Waltham, MA). Total binding
sites (Bmax) and binding affinity (Kd) were determined by a nonlinear regression
fit of bound peptides per mg of protein versus concentration of radioligand
using GraphPad Prism v 5.0 (San Diego, CA).
of 64Cu-labeled cetuximab conjugates for EGFR was determined
by a saturation binding assay based on previously published methods.25 (link) HCT116 cell membrane preparations were diluted
in binding buffer [0.1% bovine serum albumin, 50 mM Tris-HCl (pH 7.4),
5.0 mmol/L MgCl2, 0.5 μg/mL aprotinin, 200 μg/mL
bacitracin, 10 μg/mL leupeptin, and 10 μg/mL pepstatin
A], and 15 μg of membrane was added to each well of a 96-well
filtration plate (Multiscreen Durapore; Millipore; Billerica, MA).
Membranes were incubated with increasing concentrations of 64Cu-labeled cetuximab conjugates for 2 h at room temperature. Nonspecific
binding was determined by saturating receptors with excess cetuximab.
When equilibrium was reached, unbound radioactivity was filtered off
and the membranes were washed twice with 200 μL of binding buffer.
Bound radioactivity was measured with a liquid scintillation and luminescence
plate reader (1450 Microbeta; PerkinElmer; Waltham, MA). Total binding
sites (Bmax) and binding affinity (Kd) were determined by a nonlinear regression
fit of bound peptides per mg of protein versus concentration of radioligand
using GraphPad Prism v 5.0 (San Diego, CA).
6
BTV ssRNA Methyltransferase Activity Assay
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Methyl transfer from Ado[methyl-3H]Met to T7 derived BTV ssRNAs were used to determine the ability of the methylase activity of mutant VP4. A standard reaction mixture contained 50 mM TrisHCl (pH 7.5), 9 mM MgCl2, 2 mM Ado[methyl-3H]Met (87 Ci/mmol), 6 mM DTT, 1 μg of uncapped ssRNAs or 0.4 mM Cap analogue (GpppA, m7GpppG; NEB) and 2 μg purified protein. After incubation at 37 °C for 2 h, the reaction mixtures were treated with proteinase K. RNA/cap analogues were purified by phenol:chloroform extraction and precipitated with cold ethanol. Cap analogues were precipitated in 9 vol ice-cold ethanol. The transfer of the 3H-methyl was determined using PerkinElmer 1450 microbeta. As a negative control baculovirus infected cytosolic lysate was used, which was extracted from infected Sf9 cells using HNN buffer as described for VP4 recombinant proteins, treated with PEI and centrifuged to remove contaminating RNA and baculovirus particles. Reactions were performed in triplicate and data were analysed for significance using student t-test.
7
Cytotoxicity Assay for MART-1+ T Cells
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Purified MART-1 tetramer+ human T cells were co-incubated with 51Cr-labeled melanoma cells at the indicated effector:target (E:T) ratios in 96-well plates at 37°C for 4-6 hours. Lysis of tumor cells was measured by 51Cr release in the supernatant counted using a Perkin Elmer 1450 microbeta liquid scintillation & luminescence counter. Specific lysis (%) of target cells was calculated as: = [(experimental release (cpm) - spontaneous release (cpm)]/[(maximum release (cpm) - spontaneous release (cpm)] ×100%.
8
AQP4-Induced Lymphocyte Proliferation Assay
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After immunization with full‐length AQP4 protein, mice were sacrificed on day 12 after immunization and draining LN cells and splenocytes were harvested to perform peptide scan analysis. A total of 350 000 cells were plated in a 96‐well plate format together with single 20‐mer peptides with a final concentration of 10 μg/mL in clone medium (DMEM‐10% FCS supplemented with 5 × 10−5 M β‐ME, 1 mM sodium pyruvate, nonessential amino acids, l ‐glutamine and 100 U penicillin/100 μg streptomycin per milliliter). Cells were cultured for 72 h at 37°C and 5% CO2 and were pulsed with 1 μCi of 3[H] thymidine (PerkinElmer) for the last 16 h. To measure the proliferation rate, cells were harvested on glass fiber filters and analysis of incorporated 3[H] thymidine was performed in a β‐counter (1450 Microbeta; Trilux, PerkinElmer).
9
Ligand Binding Assay for Adenosine Receptors
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Isolated cell membranes were resuspended in ligand binding buffer (50 mM Tris-HCl, 10 mM MgCl2 and 1 mM EDTA, pH, 7.4) and 5 μg membrane protein/well were loaded onto poly(ethyleneimine) (0.1% v/v) treated 96-well glass fiber filter plates 49 (MultiScreen-FC filter type B, Millipore, Billerica, MA) as previously described [38 (link)]. Cells were incubated with 1.25–40 nM [3H]ZM241385 in the presence or absence of unlabeled competitor ligand (50 μM NECA) or 1.25-250 nM [3H] CGS21680 in the presence or absence of unlabeled competitor ligand (50 μM CGS21680) for 1.5 h. Once binding equilibrium was reached, membranes were washed three times with ice-cold binding buffer and then 30 μL of scintillation solution (ULTIMA gold, Perkin Elmer) was added to each well. Radioactive counts (CPMs) using a Perkin-Elmer 1450 Microbeta liquid scintillation counter were measured to determine ligand binding approximately 24 h after the addition of the scintillation solution. Non-specific binding was determined from binding to membranes in the presence of an unlabeled competitor and CPMs were subtracted from total binding to calculate specific binding.
10
Lycopene Modulates NF-κB Activity
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The NF-B activity level in cell lysates at the level of NF-B-responsive gene expression was measured by luciferase-based reporter assay (Promega dual-luciferase reporter assay system). The firefly luciferase reporter plasmid contains 5 copies of an NF-B response element (PGL4.32[luc2P/NF-B-RE/Hygro] vector; Promega). This plasmid drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). Cell treatment incubation time for luciferase assay lysates was approximately 72 h and did not include a serum starvation period. Each concentration of lycopene (0.5-5 μM) had an equivalent dilution of THF as matched control. Cells were transfected 24 h after plating with firefly and transfection control Renilla luciferase reporter plasmids using jetPRIME transfection reagents according to manufacturer's protocol (Polyplus, Nottingham, UK). Cells were incubated with transfection reagent mixed with firefly and transfection control Renilla luciferase reporter plasmids, and 72 h later were washed with PBS and lysed with luciferase assay lysis buffer (Promega). Luciferase levels were measured for 10 sec using a luminometer (1450 microbeta, PerkinElmer, Seer Green, UK). Background readings of untransfected control lysates were subtracted from luminescence values. Values were normalized against positive transfection control (Renilla luciferase).
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