BeWo cells were seeded on 24‐well plates and grown to more than 90% confluence. Cells were then washed twice with pre‐warmed uptake solution (125 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 5.6 mmol/L D‐glucose, and 25 mmol/L HEPES, pH 7.4). Cells were incubated with uptake solution containing 4 μmol/L of radioactive urate, 0.1 μmol/L of radioactive hypoxanthine or 0.02 μmol/L of radioactive estrone sulfate and their final concentrations were 200, 5, and 10 μmol/L, respectively, after adding cold molecules. Cells were incubated for indicated times at 37 and 4°C (control) and then uptake of substrates was terminated by washing cells with ice‐cold uptake solution three times. Cells were solubilized in 0.1 N sodium hydroxide and radioactivity of cell lysates was measured by a liquid scintillation counter (LSC‐5100, Hitachi Aloka Medical, Tokyo, Japan).
Lsc 5100
Manufactured by Hitachi
Sourced in Japan
The LSC-5100 is a liquid scintillation counter developed by Hitachi. It is used to measure the radioactivity of liquid samples by detecting the light emissions produced when the samples interact with scintillation materials. The LSC-5100 provides accurate and reliable measurement of alpha, beta, and gamma radioactivity in a variety of liquid samples.
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5 protocols using lsc 5100
1
Radioactive Substrate Uptake in BeWo Cells
2
Evaluation of L-alanine uptake in mice infected with EC-14
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All procedures of animal study were approved by the Animal Care Committee of Kanazawa University (AP-183983) and were conducted in accordance with the international standards for animal welfare and institutional guidelines.
EC-14 (approximately 5 × 106 CFU/100 µL) were injected into the muscle of the hind leg of mice (n = 4), described in Muranaka et al. [21 (link)]. At 2 and 8 h after infection, mice were administered 50 kBq/50 µL of 3H-L-Ala intravenously. Prior to administration, the mice were fasted for 4 h. Mice were then euthanized at 15 min (n = 4) and at 60 min (n = 4) after administration, and approximately 100 mg of blood, heart, lung, liver, kidney, spleen, infected perineal muscle, and non-infected perineal muscle (control muscle) tissues were collected.
The collected organs were divided into approximately 100 mg portions and dissolved by adding 1.0 mL of solubilizer (Solvable, PerkinElmer, Waltham, MA, USA) and crushed using a disposable homogenizer (BioMasher®, Nippi Inc., Tokyo, Japan). The radioactivity of 3H-L-Ala was measured using a liquid scintillation counter (LSC-5100, Hitachi Aloka Medical).
EC-14 (approximately 5 × 106 CFU/100 µL) were injected into the muscle of the hind leg of mice (n = 4), described in Muranaka et al. [21 (link)]. At 2 and 8 h after infection, mice were administered 50 kBq/50 µL of 3H-L-Ala intravenously. Prior to administration, the mice were fasted for 4 h. Mice were then euthanized at 15 min (n = 4) and at 60 min (n = 4) after administration, and approximately 100 mg of blood, heart, lung, liver, kidney, spleen, infected perineal muscle, and non-infected perineal muscle (control muscle) tissues were collected.
The collected organs were divided into approximately 100 mg portions and dissolved by adding 1.0 mL of solubilizer (Solvable, PerkinElmer, Waltham, MA, USA) and crushed using a disposable homogenizer (BioMasher®, Nippi Inc., Tokyo, Japan). The radioactivity of 3H-L-Ala was measured using a liquid scintillation counter (LSC-5100, Hitachi Aloka Medical).
3
Quantifying Cellular Uptake of Radiotracers
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In order to quantify the cell uptake of ions or small molecules after NIR light irradiation 111InCl3, 111In‐DTPA (Nihon Medi‐Physics Co. Ltd., Tokyo, Japan) and 3H‐H2O (PerkinElmer, Inc.) were used. 3T3‐HER2 cells were seeded at 1 × 106 on 35‐mm dishes. Cells in the Tra‐IR700 (+) group were incubated with Tra‐IR700 (5 μg/mL) for 1 hour. After washing with PBS, fresh medium or 2 different high osmotic pressure media containing these radiotracers were added to each dish. The high osmotic pressure media were prepared by adding 25 mmol/L NaCl or 50 mmol/L dextran (MW ~6000) to RPMI‐1640. Immediately after adding medium, cells were exposed to NIR light (2 J/cm2) using the LED. The cells were incubated for 3 minutes or 60 minutes at 37°C after irradiation, and they were then detached from the dishes by incubation with trypsin EDTA. After cell‐washing centrifugation with PBS, the cells were lysed in 0.1 N NaOH and radioactivity was measured with an automatic γ‐counter (1480 Wizard 3; PerkinElmer, Inc.) or a liquid scintillation counter (LSC‐5100; Hitachi‐Aloka Medical, Tokyo, Japan). Protein concentration was measured with the BCA protein assay kit (Thermo Fisher Scientific Inc.). The uptake was presented as percentage dose per milligram of protein.
4
Cellular Uptake of Methotrexate After Radiation Exposure
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Each cell was seeded at 1.0 ×105 cells/well in 12-well plastic plates. At about 24 h after seeding, cells were irradiated and divided into three groups: 4 h after irradiation; 24 h after irradiation; and control without irradiation. Each group was pre-incubated for 5 min in phosphate-buffered saline (PBS), then incubated with 3H-MTX (10 kBq/well) for 5, 10, 30, or 60 min. After incubation, cells were washed twice with 600 µL of PBS and lysed by 500 µL of .1 M NaOH. Three hundred microliters of cell lysate were mixed with 5 mL of ULTIMA GOLD (Perkin Elmer, Waltham, MA, United States) and the radioactivity of the mixture was measured using a liquid scintillation counter (LSC-5100; Hitachi Aloka Medical, Tokyo, Japan). The results are shown as the percent injected dose/number of living cells measured by an automatic cell counter (LUNA FX7™; Logo Biosystems, Gyeonggi-do, South Korea).
5
Bacterial Amino Acid Uptake Kinetics
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E. coli K-12 at 1.3 × 106 CFU/200 µL and EC-14 at 1.2 × 108 CFU/100 µL were seeded in 5 mL of amino acid-free DMEM and incubated for 1, 2, 4, 6, 8, 12, and 24 h. After incubation, 37 kBq/10 µL of 3H-L-Ala, 3H-L-Met, 3H-L-Glu, and 3H-L-His were added to the bacterial solution and incubated for 5 min at 37 °C with gentle shaking. K-12 and EC-14 were collected by centrifugation at 7000× g for 10 min at 4 °C, washed three times with 5 mL of PBS.
K-12 and EC-14 were lysed by the addition of 1 mL of 0.1 M NaOH. The radioactivity of the mixture made up of 500 µL of bacterial lysate and 5 mL of liquid scintillation cocktail (ULTIMA GOLD, Perkin Elmer, Waltham, MA, USA) was measured using a liquid scintillation counter (LSC-5100; Hitachi Aloka Medical, Tokyo, Japan).
K-12 and EC-14 were lysed by the addition of 1 mL of 0.1 M NaOH. The radioactivity of the mixture made up of 500 µL of bacterial lysate and 5 mL of liquid scintillation cocktail (ULTIMA GOLD, Perkin Elmer, Waltham, MA, USA) was measured using a liquid scintillation counter (LSC-5100; Hitachi Aloka Medical, Tokyo, Japan).
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