The effect on primary human renal mesangial cell proliferation of DS-EA was assessed by [3H]-thymidine incorporation. Quiescent cells were treated with 10 μM of angiotensin II and DS-EA, and then 1 μCi of [3H]-thymidine (methyl-[3H] thymidine, 50 Ci/mmol; Amersham, Oakville, ON, Canada) was added for 24 h. Cells were extracted three times with cold 10% trichloroacetic acid (TCA) for 5 min and solubilized for at least 30 min in 0.3 N of NaOH. After harvesting, [3H]-thymidine activity levels were measured using a liquid scintillation counter (Beckman LS 7500, Fullerton, CA, USA). Each experiment was performed in triplicate or quadruplicate.
Ls 7500
Manufactured by Beckman Coulter
Sourced in United States
The LS 7500 is a laser diffraction particle size analyzer manufactured by Beckman Coulter. It is designed to measure the size distribution of particles in a sample by analyzing the pattern of light scattered by the particles when they are illuminated by a laser beam.
Automatically generated - may contain errors
5 protocols using ls 7500
1
Angiotensin II-Induced Mesangial Cell Proliferation
2
Measuring Cell Proliferation with Tritiated Thymidine
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Quiescent cells were treated with 10 ng/ml TNF-α and SMT, respectively, and 1 μCi of [3H]-thymidine was added (methyl-[3H] thymidine, 50 Ci/mmol; Amersham, Oakville, Ontario, Canada). After 24 h of incubation, cells were washed once with 2 ml of ice-cold PBS for 10 min, extracted three times with 2 ml of cold 10% TCA for 5 min each, and solubilized for at least 30 min at room temperature in 0.2 ml of 0.3 N NaOH, 1% SDS. After neutralization with 0.2 ml of 0.3 N HCl, [3H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA). Each experiment was conducted either in triplicate or in quadruplicate.
3
Measuring Mesangial Cell Proliferation
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[3H]-thymidine incorporation was measured to determine the effect on rat mesnagial cell proliferation. Quiescent cells were treated with 25 mM glucose and Oryeongsan, respectively, and 1 μCi of [3H]-thymidine was added (methyl-[3H] thymidine 50 Ci/mmol; Amersham, Oakville, Ontario, Canada). After incubation for 24 h, cells were washed once with 2 ml of ice-cold PBS for 10 minutes, extracted three times with 2 ml of cold 10% TCA for 5 minutes each time, and solubilized for at least 30 minutes at room temperature in 0.2 ml 0.3 N NaOH, 1% SDS. After neutralizing with 0.2 ml 0.3 N HCI, [3H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA). Each experiment was performed in triplicate or quadruplicate.
4
CB-839 Inhibits BMDM Proliferation
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[3H]-Thymidine incorporation was measured to determine the effect of CB-839 on BMDM proliferation. Cells were treated with vehicle or CB-839 (1 nM to 5 μM) for 24 h, and 1 μCi/ml [3H]-thymidine (PerkinElmer; CAT No. NET027E250UC) was added. After incubation for 24 h, cells were fixed with 100% ethanol for 15 min at 4°C, precipitated with cold 10% TCA, and lysed with 0.1 N NaOH. The amount of [3H]-thymidine incorporated into DNA was measured using liquid scintillation counting (Beckman LS 7500, Fullerton, CA) and corrected for cell number.
5
Measuring Human Mesangial Cell Proliferation
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[3H]-thymidine incorporation was measured to determine the effect on human mesangial cell proliferation. The mesangial cells were plated at 2.5 × 104 cells/well in 24-well cell culture plates for 24 h, and the medium was replaced with serum-free medium containing 0.1% bovine serum albumin and the incubation continued for another 24 h. Quiescent cells were treated with 10 μM angiotensin II and DS-EA, respectively, and 1 µCi of [3H]-thymidine was added (methyl-[3H] thymidine 50 Ci/mmol; Amersham, Oakville, ON, Canada). After incubation for 24 h, cells were extracted three times with cold 10% TCA for 5 min each time and solubilized for at least 30 min in 0.3 N NaOH. After neutralizing, [3H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA, USA). Each experiment was performed in triplicate or quadruplicate. Mesangial cell index was calculated for each E-plate well by RTCA Software 1.2 (Roche Diagnosis, Meylan, France). The graphs are real-time generated outputs from the iCELLigence system.
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