Cells were seeded in either 12-well plate or 96-well plates in respective media and incubated at 37°C with 5% CO2. Next day the media were removed and test amounts of the aptamer (NUAP or CTAP), AuNP-ODNs, AuNP-NUAP-STAT3d or AuNP-CTAP-STAT3d with fluorescent tag or radiolabeled by 99mTc in Opti-MEM medium were added for various time periods. After incubation, the media containing unbound fraction was removed and cells were washed twice with PBS. For dissociating the surface bound AuNP-ODN nanoconstructs, glycine buffer (0.1M, pH 2.8) was added and the plate incubated for 5 min. After transferring to test tubes the cells were treated once in glycine buffer, glycine buffer fractions and PBS fractions were pooled to determine the surface-bound fraction. Following glycine buffer treatment the surface fractions were lysed with 0.2M NaOH/0.1% SDS and the lysates were counted using a Packard Gamma Counter. ODND and AuNP-ODN constructs were added to the cells at the final concentration as 0.5 µM. The fluorescence intensity of each lysis solution was measured by using Odyssey imager (Li-Cor Biotechnology, Lincoln NE) followed by ImageJ analysis 26 (link).
Gamma counter
Manufactured by Hewlett-Packard
Sourced in United States
The Gamma Counter is a laboratory instrument used to measure the radioactivity of samples. It can detect and quantify gamma radiation emitted by radioactive isotopes. The Gamma Counter is a versatile tool for applications in fields such as nuclear medicine, environmental analysis, and scientific research.
Automatically generated - may contain errors
Lab products found in correlation
Winnonlin 5, by Pharsight (2 mentions)
1470 wizard gamma counter, by PerkinElmer (2 mentions)
Odyssey imager, by LI COR (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Apo tf, by Merck Group (1 mentions)
Discovery w fan beam densitometer, by Hologic (1 mentions)
Liaison chemiluminescence immunoassay, by DiaSorin (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Cobas amplicor hbv monitor test, by Roche (1 mentions)
Adivia1650, by Siemens (1 mentions)
Automatic analyzer 7600, by Hitachi (1 mentions)
747 automatic analyzer, by Hitachi (1 mentions)
Bq123, by Merck Group (1 mentions)
Female cb 17 scid mice, by Charles River Laboratories (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Na125i, by PerkinElmer (1 mentions)
Raw 264.7 mouse macrophage, by Korean Cell Line Bank (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
46 protocols using gamma counter
1
Cellular Uptake of AuNP Nanoconstructs
2
Vitamin D Status Measurement Protocol
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Blood samples were processed and refrigerated. The samples were transported on the day of the survey to the designated central laboratory of Neodin Medical Institute (NMI), a laboratory certified by the Korean Ministry of Health and Welfare in Seoul, Korea. Blood samples were analyzed within 24 hours after transport. Serum 25(OH)D levels were measured by radioimmunoassay methods with a WIZARD 1470 Gamma Counter (PerkinElmer, Finland) and a Gamma Counter (Hewlett Packard, USA) in the 4th and 5th KNHANES, respectively [19 (link)]. Vitamin D deficiency was defined as a level of <20 ng/ml as per the recent guidelines proposed by the Institute of Medicine (IOM).
3
Isolation and Quantification of PrPSc in Blood Fractions
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Whole blood was diluted with 2-fold volume of phosphate buffered saline, and gently floated onto equal volume of Ficoll-Paque Premium (GE Healthcare, London). The sample was centrifuged at 400 × g for 30 minutes at room temperature. Fractions were separated into plasma, PBMC, and RBC fractions. The radioactivity from 125I-PrPSc was measured after TCA precipitation of each fraction by a gamma counter (Packard), and results were presented as %ID/ml of original whole blood volume. Representative numbers of cells in PBMC and RBC fractions were counted, and the values were 1 × 106 cells/ml and 4 × 108 cells/ml of whole blood, respectively.
4
Radiolabeled VWF Binding Assay
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Human VWF was radiolabeled with 125iodine-isotope by a standard chloramine T method and binding experiments with [125I]-VWF were performed as described previously (Bergmann et al., 2001 (link)). Briefly, 109 bacteria grown to mid-log phase were incubated with 20 nCi of radiolabeled VWF for 30 min at RT. Pneumococci were sedimented by centrifugation and after washing twice, pellet-bound radioactivity representing bound VWF was measured in a gamma counter (Packard). VWF binding was expressed as a percentage of total added radioactivity. The labelling efficiency was calculated using fetal calf serum and used to define the level of unspecific binding. The shown binding data were normalized by subtraction of this level.
5
In Vitro Activation of ProINS-Tf
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A conversion study was performed to confirm the in vitro activation of ExpressTec-ProINS-Tf and to compare the conversion rates of both ProINS-Tfs. In this study, H4IIE rat hepatoma cells (ATCC) were treated with ProINS-Tfs from rice or HEK293 cells, in the presence or absence of 1000-fold excess of apo-Tf (Sigma), followed by incubation at 37 °C for 12 h. At the indicated time points, cell culture media were collected and subjected to insulin-specific radioimmunoassay (RIA, Millipore) that has less than 0.2% cross-reactivity with human proinsulin. The insulin concentrations of media were determined by insulin-specific RIA according to the manufacturer’s instructions, and the 125I radioactivity was counted using a gamma counter (Packard, Downers Grove, IL, USA).
6
Measuring Bone Health: Vitamin D, PTH, and BMD
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Laboratory blood tests included 25-OH vitamin D3, PTH, and ALP levels. 25-OH Vitamin D3 was measured by radioimmunoassay using a gamma counter (Hewlett Packard, Palo Alto, CA, USA). PTH was measured using a LIAISON chemiluminescence immunoassay (Diasorin, Stillwater, MN, USA), and ALP was measured using an enzyme assay using a Hitachi Automatic Analyzer 7600 (Hitachi, Tokyo, Japan) and external and internal quality controls.
Bone mineral density (BMD) (g/cm2) was measured by DEXA scan (DISCOVERY-W fan-beam densitometer; Hologic Inc., Bedford, MA, USA). The accuracy of BMD in KNHANES was assessed by educated affiliated osteoporosis assessors, and interpretation of the BMD results was corrected and standardized by quality control [24 (link)].
BMD was estimated at lumbar vertebrae (L1–4) and the femur (total femur and femoral neck). The T-score, which is a reference standard deviation value, compared with BMD of a healthy 30-year-old, was used to compare BMD between groups. Osteoporosis was defined when the T-score was ≤ −2.5, and a T-score > −2.5 but < −1 was classified as osteopenia [25 (link)].
Bone mineral density (BMD) (g/cm2) was measured by DEXA scan (DISCOVERY-W fan-beam densitometer; Hologic Inc., Bedford, MA, USA). The accuracy of BMD in KNHANES was assessed by educated affiliated osteoporosis assessors, and interpretation of the BMD results was corrected and standardized by quality control [24 (link)].
BMD was estimated at lumbar vertebrae (L1–4) and the femur (total femur and femoral neck). The T-score, which is a reference standard deviation value, compared with BMD of a healthy 30-year-old, was used to compare BMD between groups. Osteoporosis was defined when the T-score was ≤ −2.5, and a T-score > −2.5 but < −1 was classified as osteopenia [25 (link)].
7
Chromium Release Cytotoxicity Assay
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The CMC assay has been described in detail previously [44] (link). Targets were labeled with 100 μCi 51Cr (Na251CrO4; ICN, Irvine, CA) for 2 hours at 37°C, washed 4 times and cultured for 12 hours at 37°C with 104 lymphocytes (effector∶target ratio of 10∶1). Supernatants were removed and counted in a Packard Gamma Counter. Pellets were lysed using 6N HCl and the pellets were removed and counted. To determine maximal releasable 51Cr, some wells were directly treated with HCl to lyse all targets. Percent 51Cr release was calculated as: CPM in supernatant/(CPM in supernatant + CPM in pellet) ×100 for each well. Percent specific lysis represents: (% 51Cr release in experimental wells - % 51Cr release in medium control wells)/(% 51Cr in HCl lysed maximum release wells - % 51Cr release in medium control wells) ×100.
8
Metabolic Regulation by SLC7A2
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To assess the role of SLC7A2 in glucagon and insulin secretion, Slc7a2+/+, Slc7a2+/− and Slc7a2−/− mice were fasted for 6 hours and then injected intraperitoneally with glucose, arginine, or both to final concentrations of 2g / kg body weight each. Blood was collected retroorbitally before injection (Fasting) and 15 minutes after injection (Stimulated). Blood glucose was measured with a hand-held glucometer (Accu-Check Aviva) and remaining whole blood was spun, serum collected into separate tubes and stored at −80°C for glucagon and insulin analyses.
Serum hormones were analyzed in the Vanderbilt Hormone and Analytical Services core. Serum glucagon was analyzed in a two-site enzyme sandwich ELISA (Mercodia). Serum insulin was analyzed by dual antibody radioimmunoassay and counted in a Packard Gamma counter.
Serum hormones were analyzed in the Vanderbilt Hormone and Analytical Services core. Serum glucagon was analyzed in a two-site enzyme sandwich ELISA (Mercodia). Serum insulin was analyzed by dual antibody radioimmunoassay and counted in a Packard Gamma counter.
9
Somatostatin Receptor Internalization Assay
10
Quantification of Hepatitis B Surface Antigen
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IRMA kits (RIAKEY; Shin Jin Medics, Goyang, Korea) were used for HBsAg quantification according to the manufacturer’s instructions. Briefly, the samples were incubated with primary antibody-coated beads for 1 hour. The beads were then removed by washing 4 times in washing solution, and then treated with 125I-conjugated secondary antibody for 30 minutes. Next, radioactivity was measured in counts per minute (CPM) by using a gamma counter (Packard, Downers Grove, IL, USA). Serum HBsAg titers were determined by reading the CPM values off a standard curve. The test was considered positive when the serum HBsAg levels exceeded 0.1 IU/mL (detection range, 0.05 to 250 IU/mL). When HBsAg levels exceeded the detection range, the test was repeated after diluting the sample. A COBAS Am-plicor HBV Monitor test (Roche Molecular Systems, Pleasanton, CA, USA) was used to measure serum HBV DNA concentrations during the study period, which has a lower detection limit of 20 IU/mL.
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