These were performed as described earlier [32 ]. MC3T3 cells (1×104 cells/well) were seeded in 24-well plates. Upon confluence, ascorbic acid (100 μg/mL) was added. After 72 hours, 5mM β-glycerophosphate, 45CaCl2 (18.5 kBq/well), and either vehicle (PBS), prostacyclin (10μM), or carbaprostacyclin (10μM) were added. Vehicle, prostacyclin (10μM) or carbaprostacyclin (10μM) were added daily, and media were changed every 3.5 days. At days 7 and 14 post-addition of β-glycerophosphate, cells were rinsed with PBS, and frozen at −20 °C. Frozen cells were lysed with 500μL of 10mM Tris-HCl (pH 7.4), 10mM MgCl2, and 0.1% (w/v) Triton X-100, and centrifuged at 14,000 x g for 5 minutes. Alkaline phosphatase activity was measured by fluorescence assay (QuantiFluo, BioAssay Systems) in the supernatant. Radioactivity (45Ca) was measured with a TopCount NXT (Perkin Elmer) scintillation counter by adding 50μL of the cell lysate to 150μL of scintillant (Microscint 40).
Topcount nxt
Manufactured by PerkinElmer
Sourced in United States, Japan, United Kingdom
The TopCount NXT is a high-performance microplate reader designed for rapid and accurate detection of luminescent, fluorescent, and radiometric assays. It features a modular design and supports a wide range of microplate formats, allowing it to be configured to meet the specific requirements of various research and diagnostic applications.
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Lab products found in correlation
3h thymidine, by PerkinElmer (15 mentions)
Filtermate harvester, by PerkinElmer (5 mentions)
Pipetting robot, by Beckman Coulter (4 mentions)
Decorin, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Lumaplate, by PerkinElmer (4 mentions)
Anti mouse cd28, by Thermo Fisher Scientific (3 mentions)
F ab 2 fragment goat anti mouse igm, by Jackson ImmunoResearch (3 mentions)
Anti mouse cd3, by BD (3 mentions)
Concanavalin a (cona), by Merck Group (3 mentions)
Filter plate, by PerkinElmer (3 mentions)
96 well plate, by BD (3 mentions)
Ovalbumin (ova), by Merck Group (2 mentions)
Gf b filter, by Cytiva (2 mentions)
D luciferin potassium salt, by GoldBio (2 mentions)
Unifilter 96, by PerkinElmer (2 mentions)
Microscint 20, by PerkinElmer (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Soluble anti cd28 1 μg ml antibody, by Thermo Fisher Scientific (2 mentions)
Topseal a plus, by PerkinElmer (2 mentions)
97 protocols using topcount nxt
1
Osteoblast Mineralization Assay with Prostacyclins
2
Quantifying Tumor-Induced Immune Suppression
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Single-cell suspensions from spleen were stained and sorted on BD FACS Aria (BD Biosciences). CD11b
+Ly6G
+Ly6C
−PMN
TUMORwere plated in dishes in 1,640 RPMI with 10% FBS and co-cultured at different ratios with splenocytes from OT-itransgenic mice in the presence of 0.1 ng/mL ovalbumin (Sigma–Aldrich). After 48 hours, cells were incubated with (3H) thymidine (PerkinElmer, Waltham, United States) for 16 to 18 hours. Proliferation was quantized by using the TopCount NXT instrument (PerkinElmer).
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Single-cell suspensions from spleen were stained and sorted on BD FACS Aria (BD Biosciences). CD11b
+Ly6G
+Ly6C
−PMN
TUMORwere plated in dishes in 1,640 RPMI with 10% FBS and co-cultured at different ratios with splenocytes from OT-itransgenic mice in the presence of 0.1 ng/mL ovalbumin (Sigma–Aldrich). After 48 hours, cells were incubated with (3H) thymidine (PerkinElmer, Waltham, United States) for 16 to 18 hours. Proliferation was quantized by using the TopCount NXT instrument (PerkinElmer).
3
Determining Cytotoxic T-Cell Specificity and Avidity
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The specific lytic activity of the NY-ESO-1 CD8 T-cell lines or Melan-A specific T cell clones was assessed by presentation of HLA-A*02 peptide variants of NY-ESO-1157-165 and Melan-A26-35 on 51Cr-labeled TAP-deficient T2 cells. T2 cells were labeled with 51Chromium (Amersham Biosciences) for 1h, washed and resuspended in culture medium. 51Cr-labeled cells were then incubated for 4h at 37°C with effector T cells at an Effector : Target ration of 10:1 with titrated amount of NY-ESO-1157-165 and Melan-A26-35 peptide variants At the end of the incubation time, the supernatants were harvested and radioactivity was counted in an automatic gamma-counter TopCount NXT (Perkin-Elmer). The percentage of specific lysis was determined using the formula: (experimental-spontaneous release)/(maximum-spontaneous) x 100. Internal controls were included in each assay to measure the spontaneous release (target cells alone) and the total release (target cells with 1 M HCl) (49 (link)).. Functional avidity (EC50 = peptide concentration giving 50% maximal killing) was derived from the nonlinear log(agonist) vs. response equation using Prism software (GraphPad, v.9.1.1).
4
Caspase-3/7 Activity Quantification
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Caspase-3/7 activity was determined using Promega’s caspase-3/7 assay according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, cells were transferred in a white opaque 96-well plate, incubated for 1 h with Caspase-Glo reagent and the enzymatic activity of caspase-3/7 was measured using a TopCount NXT microplate luminescence counter (PerkinElmer, Waltham, MA, USA). To enable normalisation of data to total cellular protein content, the sulforhodamine B (SRB) assay was performed in parallel for all samples.27 (link)
5
Arg-Dependent T and B Cell Activation
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Splenic lymphocytes (2 × 105 cells/well) from naïve Arg+/+ and Arg−/− mice were stimulated in vitro with purified anti-mouse CD3 (BD Biosciences, San Jose, CA) and anti-mouse CD28 (eBioscience, San Diego, CA), Concanavalin A (ConA) (Sigma-Aldrich, St. Louis, MO), lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO), and F(ab’)2 Fragment Goat Anti-Mouse IgM (Jackson Immuno Research, West Grove, PA). Cells were stimulated for 72 hours, and pulsed with [3H]- thymidine (1 μCi/well) (Perkin Elmer, Waltham, MA) for the last 16–18 hours. Cells were harvested and [3H]-thymine incorporation monitored on a TopCount NXT (Perkin Elmer, Waltham, MA).
6
Recombinant Human ACL Activity Assay
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The activity of recombinant human ACL was carried out essentially as described in ref. 63 (link). Briefly, 7.5 × compounds were added to a 96-well PolyPlate containing 60 μl of buffer (87 mM Tris, pH 8.0, 20 μM MgCl2, 10 mM KCl and 10 mM DTT) per well with substrates CoA (200 μM), ATP (400 μM) and [14C]-citrate (specific activity: 2 μCi μmol−1)(150 μM). Reaction was started with 4 μl (300 ng per well) ACL and the plate incubated at 37 °C for 3 h. The reaction was terminated by the addition of 3.5 μl 500 mM EDTA. 200 μl MicroScint O was then added to the reaction mixture and incubated at room temperature overnight with gentle shaking. The [14C] acetyl-CoA signal was detected (5 min per well) in a TopCount NXT liquid scintillation counter (Perkin-Elmer, Waltham, MA).
7
Measuring Prokaryotic Ribosome Frameshifting
8
DNMT1 Enzymatic Assay Protocol
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His-DNMT1 (182 kDa, human) was cloned, expressed and purified as described by Lee et al. [20] (link). The DNMT1 assay was performed according to Gros et al. [21] (link). Briefly, the reaction was started by addition of 90 nM of DNMT1 on a mix containing the tested compound (up to 1% DMSO), 1 µM of a AdoMet/[methyl-3H]-AdoMet mix in a ratio of 3-to-1 (isotopic dilution 1*:3) and 0.3 µM of biotinylated DNA duplex in 10 µL final volume. The reaction was incubated at 37°C for 2 h in reaction buffer (20 mM HEPES pH 7.2, 1 mM EDTA, 50 mM KCl, 25 µg/mL BSA). 8 µL are then transferred into a streptavidin-coated Flashplate PLUS (PerkinElmer) containing 190 µL of 20 µM AdoHcy (Sigma-Aldrich) in 50 mM Tris-HCl pH 7.4. The Flashplate was agitated at room temperature for 1 h, washed three times with 200 µL of 0.05% Tween-20 in 50 mM Tris-HCl pH 7.4 and read in 200 µL of 50 mM Tris-HCl pH 7.4 on TopCount NXT (PerkinElmer). Percentages of inhibition were calculated with the same formula as in DNMT3A assay.
9
Luciferase Assay for Renin-dCas9 Regulation
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The luciferase assays were performed with HEK_dCas9-SAM_Renin-luciferase cells. The cells were transfected with all possible combinations of the five guides as described above. The sample size of each combination was n = 6. Immediately after transfection, the 96-well plates were sealed and the luciferase activity was measured over time at 37°C in the TopCount® NXT (Perkin Elmer, Waltham, USA). Luciferase activity is expressed in counts per second (cps).
10
Plasmodium falciparum Inhibition Assay
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The 50% inhibitory concentrations (IC50) determination test was carried out by isotopic 42 h 3H-hypoxanthine incorporation assays as previously described [34 (link)] with minor modifications. Briefly, P. falciparum cultures at ring-stage were highly synchronized by two consecutive treatments with 5% sorbitol in PBS (v/v) at 40-h intervals and diluted down to 0.3–0.5% parasitemia and 2% hematocrit. Parasites were dispensed into 96-well plates containing 14 serially diluted concentrations of drug ranging from 0 to 50 µM for GA 1 , AZC-GA 5 and cpd. 7 , and from 0 to 952 nM for dihydroartemisinin, and incubated as described above in the presence of 5% 3H-hypoxanthine (Perkin Elmer, Hopkinton, MA, USA) for 42 h. 3H-hypoxanthine uptake was then evaluated by scintillation counting (Top Count NXT, Perkin Elmer) and results were expressed as the inhibitory concentrations IC50, defined as drug concentrations at which 50% of 3H-hypoxanthine incorporation was inhibited compared with drug-free controls. IC values were established by non-linear regression with ICEstimator software (http://www.antimalarial-icestimator.net/ ) [35 (link),36 (link)]. All tests were done in triplicates.
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