Albumin diagnostic kit

Broncho-alveolar lavage (BAL) samples were collected following previously established protocols [12 (link)]. In brief, the trachea was cannulated using a catheter, followed by two consecutive lavages with sterile PBS. The BAL fluid was centrifuged at 900× g for 10 min, and the supernatant was stored at −80 °C for later analysis.
To assess lung injury, total protein and albumin concentrations and lactate dehydrogenase (LDH) activity were measured in BAL samples. Proteins were quantified by the bicinchoninic protein assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) as described previously [12 (link)]. Albumin levels were quantified using a colorimetric assay based on bromocresol green binding, employing an Albumin Diagnostic Kit (Wiener Lab, Buenos Aires, Argentina). LDH activity was measured as the production of NADH, following the procedures and reagents provided by Wiener Lab. Briefly, 20 µL of BAL samples were incubated at 30 °C with 1 mL of reactive A: Tris HCl (80 mM, pH 7.2), pyruvate (1.6 mmol/L), NADH (0.2 mmol/L), and NaCl (200 mmol/L). The initial absorbance (340 nm) and the absorbance at 1, 2, and 3 min were read. The average absorbance difference/min (∆A/min) was determined, and the results are shown in units per liter of BAL fluid, following the table correction provided by the kit.
Neutrophils counts in BAL samples were also assessed as a measure of inflammation. The total leukocyte counts in BAL samples were measured using a hemocytometer, while differential counts were performed with May Grünwald–Giemsa stain [12 (link)]. Total leukocyte counts and neutrophil percentages in smears were used to calculate the absolute number of BAL neutrophils.

Albumin content, as a measure to quantify the increased permeability of the alveolar-capillary barrier, and the activity of the intracellular enzyme lactate dehydrogenase, an indicator of cellular cytotoxicity, were determined in BAL samples [22 (link),23 (link)]. Albumin content was determined colorimetrically based on albumin binding to bromocresol green using a Wiener-Lab albumin diagnostic kit. LDH activity, expressed as units per liter of BAL, was determined by measuring the formation of the reduced form of NAD⁺ using Wiener’s reagents and procedures (Wiener-Lab).

The albumin content was used as a measure to quantify the increased permeability of the alveolar–capillary barrier, and the activity of the intracellular enzyme lactate dehydrogenase, an indicator of cellular cytotoxicity, was determined in BAL samples [59 (link)]. The albumin content was determined colorimetrically based on albumin binding to bromocresol green using a Wiener-Lab albumin diagnostic kit. LDH activity, expressed as units per liter of BAL, was determined by measuring the formation of the reduced form of NAD⁺ using Wiener’s reagents and procedures (Wiener-Lab, Rosario, Argentina).
The lung wet:dry weight ratio was measured as previously described [37 (link),38 (link)]. Briefly, mice were euthanized and exsanguinated, and their lungs removed, weighed, and dried in an oven at 55 °C for 7 days. After drying, the lungs were weighed again. The wet:dry weight ratio was then calculated as an index of intrapulmonary fluid accumulation, without correction for blood content.