For protein and cellular analysis, the BAL components were separated by centrifugation. The protein concentration inside the BAL fluid was analyzed by a protein assay (PierceTM BCA Protein Assay, Fisher Scientific, Hampton, VA, United States) based on the manufacturer’s instructions. Albumin concentration in the BAL was measured via ELISA (Rat Albumin ELISA Kit, Bethyl Laboratories, Montgomery). The BAL samples were diluted 1:1000 and compared to the standard curve according to the manufacturer’s protocol. For cellular analysis, the BAL cells were separated from the rest of the BAL via centrifugation. Cytospin slides (Thermo Shandon Cytospin 3, Marshall Scientific, Hampton VA, United States) were prepared with 50.000 cells per BAL diluted in 100 μl phosphate buffer (PBS). Lymphocytes, macrophages and neutrophilic granulocytes were differentiated after staining the Cytospins with DiffQuik. Using Visiopharm® Software a systematic uniform area sampling was performed and at least 100 to 200 cells were categorized and counted using a primary magnification of 40×. Furthermore, we assessed the concentration of IL-6 within the BAL using an ELISA Kit (DuoSet® ELISA Rat IL-6, R&D Systems, Minneapolis, MI, United States). We utilized 96 well-plates and photometric measurements for cytokine concentration. Calculations were made with Magellan Software.
Rat albumin elisa kit
Manufactured by Fortis Life Sciences
Sourced in United States
The Rat Albumin ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of rat albumin in biological samples.
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Lab products found in correlation
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Thermo shandon cytospin 3, by Marshall Scientific (1 mentions)
Detectx urinary creatinine kit, by Arbor Assays (1 mentions)
Quantichrom urea assay kit, by BioAssay Systems (1 mentions)
Mtp 810, by Corona Electric (1 mentions)
Protein carbonyl colorimetric assay, by Cayman Chemical (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
8 protocols using rat albumin elisa kit
1
Comprehensive BAL Fluid Analysis
2
Urinary Biomarkers Quantification
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Urinary creatinine concentration was determined using a colorimetric assay, according to the manufacturer’s instructions (DetectX urinary creatinine kit, Arbor Assays, MI, USA). Urinary albumin concentration was determined using a rat albumin ELISA kit, according to the manufacturer’s instructions (Bethyl Laboratories, Inc, TX, USA). Urinary neutrophil gelatinase-associated lipocalin (NGAL) concentration was determined using a rat Lcn2 ELISA kit, according to the manufacturer’s instructions (RAB0906, Sigma Aldrich, UK).
3
Urine Albumin and Creatinine Analysis
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Urine albumin was estimated using a rat albumin ELISA kit (Bethyl Laboratories, TX, USA). Urine creatinine was analyzed using a modified Jaffe’s method (Randox, Crumlin County Antrium, UK).
4
Quantification of Albumin and Urea in Hepatocytes
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The amount of albumin in culture medium was measured using an enzyme-linked immunosorbent assay kit (rat albumin ELISA kit, Bethyl Laboratories, Montgomery, TX). The amount of urea in culture medium was measured using an assay kit (QuantiChrom urea assay kit, BioAssay Systems, Hayward, CA). For both assays, respective absorbency at 450 nm or at 430 nm was measured using a microplate reader (MTP-810, Corona Electric, and Hitachi, Japan). All the values were normalized by the number of hepatocytes and the data were expressed as protein amount 10–6 cells time−1.
5
Quantifying Urinary Albumin and Creatinine
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Urine albumin level was evaluated using a rat albumin ELISA kit (Bethyl Laboratories, TX, USA) and urine creatinine level was assessed using a modified Jaffe’s method (Randox, Crumlin County Antrium, UK).
6
Quantifying Oxidative Stress Biomarkers
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Thiobarbituric acid reactive substances (TBARS) was measured in urine and tissue homogenate of kidney cortex by adding 50 μl sample to 42 μl 0.67% thiobarbituric acid. Samples were vortexed and heated to 97°C for 60 min. After cooling the samples on ice, 50 μl methanol:1 mM NaOH (91:9) was added, the samples vortexed and centrifuged at 3000 rpm for 5 min at room temperature. The supernatant was analyzed for fluorescence using excitation/emission of 532/553 nm and the concentration calculated using a standard curve of malondialdehyde. All values were corrected for protein concentration. Concentration of TBARS in urine samples were multiplied by urine flow to get urinary TBARS excretion. Protein content was determined by DC-Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Albuminuria was measured by rat albumin ELISA kit, (Bethyl Laboratories, Montgomery, TX, USA) according to manufacturer´s instruction. Protein carbonylation in kidney cortex was determined spectrophotometrically by protein Carbonyl Colorimetric Assay (Cayman Chemicals, MI, USA) and normalized for protein concentration.
7
Evaluating Renal Function and Injury Biomarkers
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Inulin clearance (mL/min): renal function was evaluated based on inulin clearance. Inulin was injected in the right jugular vein, with a loading dose of 100 mg/kg, followed by a continuous infusion of 0.04 mL/min. After a 30 min stabilization period, three urine samples were collected through the bladder catheter and two blood samples were then collected through the carotid catheter. The serum and urine inulin were measured using the anthrone method [17 (link), 18 (link)]
Urinary neutrophil gelatinase-associated lipocalin (NGAL, pg/mL): urinary NGAL was determined using the Rat NGAL ELISA Kit, BioVendor, research and diagnostic products
Serum and urinary creatinine (Cr, mg/dL): serum and urinary creatinine were measured using the Jaffe method. The results were expressed in mg/dL; oxidative parameters were corrected using urinary creatinine [19 (link)]
Urinary albumin (mg/24 h): albuminuria was determined using the Rat Albumin ELISA Kit, Bethyl Laboratories
8
Quantifying Serum Creatinine and Albuminuria
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Serum creatinine was measured using The DetectX Serum Creatinine kits from Arbor Assays (Ann Arbor, MI). Albuminuria was determined by a Rat Albumin ELISA Kit from Bethyl Laboratories (Montgomery, TX).
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