To obtain the BALF, the lungs were lavaged three times with ice-cold PBS (0.5 ml) and withdrawn each time using a tracheal cannula (a total volume of 1.5 ml). The collected BALF was centrifuged at 1000×g for 15 min at 4 °C, and the supernatants were collected and frozen at − 80 °C for subsequent assays. The cell pellet was resuspended in PBS, and after excluding the dead cells by trypan blue staining, the total number of inflammatory cells in the BALF was determined by counting the cells with a haemocytometer (Beckman Coulter, Inc). To analyse the cell numbers, 100 μl of BALF was centrifuged onto slides by a Cytospin (Thermo Fisher Scientific, Waltham, USA). After the slides were dried, the cells were fixed and stained using Wright Stain solution (32857, Sigma, USA) according to the manufacturer’s instructions. The number of polymorphonuclear neutrophils (PMNs) was classified by a laboratory technologist blinded to the experimental design to determine the percentage of neutrophils. The frozen BALF supernatant was thawed and thoroughly mixed, and the total protein concentration was determined by the BCA (bicinchoninic acid) method.
Haemocytometer
Manufactured by Beckman Coulter
Sourced in United States
A Haemocytometer is a laboratory device used to count and determine the concentration of cells, such as blood cells, in a sample. It consists of a thick glass slide with a mirrored counting chamber engraved with a calibrated grid pattern. Samples are placed on the grid, and the cells are counted manually under a microscope to determine their number and density.
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3 protocols using haemocytometer
1
Bronchoalveolar Lavage Fluid Analysis
2
Investigating Vanadium Complex Effects on Diabetic Rats
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Post-induction, the short-term haemanetic effects of the novel vanadium complex (40 mg kg – 1 p.o) were investigated in STZ-induced diabetic rats. The experimental animals were divided into the following groups; non-diabetic (ND) diabetic control (DC), novel vanadium complex (40 mg kg −1 p.o) (VAN) and insulin (0.175 mg/kg-1, s.c) (INS) treated animals that served as positive control. All animals (n=6 in each group) were housed individually in Makrolon polycarbonate metabolic cages (Tecniplast, Labotec, South Africa). The vanadium complex was administered twice every third day at 09h00 AM and 15h00PM by means of an 18-gauge gavage needle (Kyron Laboratories (Pty) LTD, Benrose, South Africa). The diabetic group which received DMSO/saline (3mL kg-1, P.O.) and insulin group (0.175 mg kg-1 s.c) acted as a negative control and positive control, respectively. Over the period of 5 weeks the RBC count, haematocrit (Hct), haemoglobin (Hb), MCV, MCH, MCHC and blood glucose concentrations, were monitored every third day using a haemocytometer (Beckman Coulter, Indianapolis, United States) and OneTouch select glucometer (Lifescan, Mosta, Malta, United Kingdom) respectively.
3
Measuring Hematological and Hormonal Changes
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At the end of the 5-week experimental period, all the animals were sacrificed by exposing them to halothane via a gas anaesthetic chamber (100 mg kg-1) for 3 minutes (Biomedical Resource Unit, UKZN, and Durban, South Africa). Thereafter, blood (100 µL) was collected by cardiac puncture (RBCP) into individual pre-cooled EDTA tubes to assess the effects of novel vanadium complexes on red blood cells, mean corpuscular volume, mean corpuscular haemoglobin concentration and haematocrit using a haemocytometer (Beckman Coulter, Indianapolis, United States). Thereafter, more blood was collected via cardiac puncture into pre-cooled heparinize containers and centrifuged (Eppendorf centrifuge 5403, Germany) at 4 °C, 503 g for 15 minutes for hormonal analysis. The kidneys were removed and weighed before freezing in liquid nitrogen. Thereafter, the kidneys were stored in an Ultra Bio Freezer (Snijders Scientific, Tilburg, Netherlands) at −80°C.
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