Prior to siRNA transfection, the media was changed on 5-days cultured MDMs to Opti-MEM reduced-serum medium (Gibco) and the cells left for approximately 1 h. MDMs were then transfected using Lipofectamine RNAiMAX (ThermoFisher Scientific) with either SMARTpool: ON-TARGETplus human CDK9 siRNA or ON-TARGETplus Non-targeting Control Pool siRNA, both 50 nM final concentration (Dharmacon, Horizon Discovery). MDMs were left to transfect for 3 days, after which they were stimulated for 24 h with 100 ng/ml LPS and supernatants were collected for cytokine analysis by ELISA. LDH was measured using a colorimetric assay kit (Roche, United Kingdom) and results were compared to a positive control (supernatants from MDM lysed using 2% Triton-X 100).
Colorimetric assay kit
Manufactured by Roche
Sourced in United States
Colorimetric assay kits are laboratory reagents used to quantitatively measure the concentration of specific analytes in a sample through color-based detection. These kits provide a standardized and reproducible method for colorimetric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mouse metabolic hormone magnetic bead panel, by Merck Group (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
On targetplus non targeting control pool sirna, by Horizon Discovery (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
Specific elisa kits, by Nanjing Jiancheng (1 mentions)
Bicinchoninic acid assay kit, by Merck Group (1 mentions)
Quantichrom assay kit, by BioAssay Systems (1 mentions)
Mouse high sensitivity t cell magnetic bead panel, by Merck Group (1 mentions)
Mouse testosterone elisa kit, by Abcam (1 mentions)
Cobas 8000 c702 chemistry analyzer, by Roche (1 mentions)
9 protocols using colorimetric assay kit
1
Knockdown of CDK9 in Macrophages
2
Trypan Blue Staining and LDH Assay for NSCs
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For trypan blue staining, 10 μl aliquots of cells were incubated with 10 μl of trypan blue solution for 2 m. Unstained live cells were counted with a hemocytometer. A colorimetric assay kit (Roche Boehringer–Mannheim, IN, USA) was used to quantify LDH released from cultured NSCs according to the manufacturer’s instructions. Cell viability was assessed using an ELISA plate reader (Synergy H1 Hybrid reader, BioTek Instruments, Winooski, VT, USA) at 490 nm with a reference wavelength of 690 nm. All results were normalized to the OD of an identical well without cells41 (link).
3
Kidney Function Biomarkers Analysis
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At the end of the 4-week infusion period, blood samples were collected from the heart, and the heart and kidney were harvested and weighed. Serum creatinine was quantified using the colorimetric assay kit (Roche Diagnostics). The concentrations of the proteins and albumin were measured in the collected urinary samples using specific ELISA kits (Jiancheng, Nanjing) according to the manufacturer's instructions. Albumin excretion is presented as micrograms of albumin per milligram of creatinine.
4
Kidney Injury Biochemical Analysis
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At the termination of experiment, right kidneys were immediately removed en bloc and snap-frozen in liquid nitrogen for biochemical analyses. The whole kidney was homogenized. Renal tissue lactate levels were determined by a nicotinamide adenine dinucleotide enzyme coupled colorimetric assay as previously described (18 (link)). Gelatinolytic activities of MMP-2 and MMP-9 were quantified using gelatin zymography as previously described (21 (link)). Tissue protein content was quantified using a bicinchoninic acid assay kit (Sigma-Aldrich Canada Ltd., Oakville, ON).
Both plasma and urine creatinine (Cr) levels were measured using a commercially available QuantiChrom assay kit (DICT-500; Bioassay Systems, Hayward, CA). N-Acetyl-D-glucosaminidase (NAG) activity was measured in the urine sample by a commercially available colorimetric assay kit (no. 875406; Roche, Indianapolis, IN) and normalized to urine Cr level.
Both plasma and urine creatinine (Cr) levels were measured using a commercially available QuantiChrom assay kit (DICT-500; Bioassay Systems, Hayward, CA). N-Acetyl-D-glucosaminidase (NAG) activity was measured in the urine sample by a commercially available colorimetric assay kit (no. 875406; Roche, Indianapolis, IN) and normalized to urine Cr level.
5
Comprehensive Metabolic and Hormonal Analysis
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After 8 weeks of GABA treatment, blood samples were obtained through cardiac puncture and centrifuged at 3000× g for 20 min at 4 °C, and serum samples were stored at −80 °C until subsequent analysis. Serum insulin concentrations were measured using a Mouse Metabolic Hormone Magnetic Bead Panel (Merck Millipore, Burlington, MA, USA). Serum concentrations of TG, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol were determined using colorimetric assay kits (Roche). Serum concentrations of TNF-α and IL-1β were measured using a Mouse High-sensitivity T Cell Magnetic Bead Panel (Merck Millipore, Burlington, MA, USA). Serum testosterone concentrations were measured using a Mouse Testosterone ELISA Kit (Abcam, Cambridge, UK).
6
Serum Biomarker Analysis in Mice
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Blood samples were obtained by cardiac puncture under terminal anesthesia and centrifuged at 3000× g for 20 min at 4 °C to collect serum. The serum concentrations of insulin were measured using a mouse metabolic hormone magnetic bead panel (Merck Millipore, Burlington, MA, USA). The serum concentrations of insulin, TG, total cholesterol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol, and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were determined using colorimetric assay kits (Roche).
7
Serum Biomarker Analysis in Mice
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After 6 weeks, blood was collected by cardiac puncture immediately after euthanasia and serum was separated by clotting for 30 min at 4 °C and centrifugation at 2000× g for 20 min. The serum concentrations of TG, FFA, cholesterol, aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) activities were determined using colorimetric assay kits (Roche, Basel, Switzerland). Serum cholesterol, TG, FFA, AST, ALT, and LDH were measured using Cobas 8000 c702 Chemistry Analyzer (Roche, Basel, Switzerland).
8
Serum Lipid and Enzyme Profiling
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Blood samples were collected at 09:00 am by cardiac puncture under terminal anesthesia. After the blood clotted for 30 min, the serum was separated by centrifugation at 4 °C and 3000× g for 20 min. The serum concentrations of triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol, and the activities of aspartate transaminase (AST) and alanine transaminase (ALT), were determined using colorimetric assay kits (Roche, Basel, Switzerland).
9
Investigating OKC's Effects on Adipocyte Differentiation
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Blood samples were obtained by cardiac puncture under terminal anesthesia and centrifuged at 3000g for 20 min at 4 °C to collect serum. The serum concentrations of insulin were measured using a Mouse Metabolic Hormone Magnetic Bead Panel (Merck Millipore, Burlington, MA, USA). The serum concentrations of TG, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol; and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using colorimetric assay kits (Roche).
Cell culture 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum (BS) and 1% penicillin/streptomycin (P/S) until confluent. After 2 days (D0), this medium was replaced with DMEM containing 10% fetal bovine serum (FBS), 1% P/S, and MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 4 μg mL -1 insulin). On D2, this medium was replaced with DMEM containing 10% FBS, 1% P/S, and 4 μg mL -1 insulin, and this was replaced every 2 days until day 8 (D8). For OKC treatment, 2-day confluent 3T3-L1 cells were incubated with different doses of OKC (0, 12, 25, 50, or 100 μg mL -1 ) every 2 days during differentiation and until mature adipocyte formation (D8).
Cell culture 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum (BS) and 1% penicillin/streptomycin (P/S) until confluent. After 2 days (D0), this medium was replaced with DMEM containing 10% fetal bovine serum (FBS), 1% P/S, and MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 4 μg mL -1 insulin). On D2, this medium was replaced with DMEM containing 10% FBS, 1% P/S, and 4 μg mL -1 insulin, and this was replaced every 2 days until day 8 (D8). For OKC treatment, 2-day confluent 3T3-L1 cells were incubated with different doses of OKC (0, 12, 25, 50, or 100 μg mL -1 ) every 2 days during differentiation and until mature adipocyte formation (D8).
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