Multiskan go

Display of the accumulated intracellular lipid droplets by adipocytes was carried out with common Oil Red O staining protocols. Briefly, cells were cultured in 6-well plates and differentiated into adipocytes as previously described. Following 10 days of differentiation, wells were aspirated and washed with PBS followed by cell fixation via addition of 10% fresh formaldehyde (in PBS, v/v). Fixation was continued for 1 h at room temperature. Staining of the lipid droplets was performed by the addition of 1 mL Oil Red O solution (prepared in 6 parts of isopropanol and 4 parts of water) into aspired and washed (with PBS) wells. After 1 h, Oil Red O staining solution was removed, and wells were air-dried. Stained lipid droplets were visualized under an optical microscope (Olympus, Tokyo, Japan). Stain from the lipid droplets was eluted by the presence of 100% isopropyl alcohol in wells. Quantification was carried out by colorimetry measuring the absorbance of the wells (containing retained dye and 100% isopropyl alcohol) at 500 nm using a microplate reader (MultiSkan GO).

The levels of apelin in the serum of healthy controls and silicosis patients or mice were detected by Human Apelin ELISA kits or Mouse Apelin ELISA kits (Novus Biologicals, USA), respectively. This ELISA kit has cross-reactivity with apelin isoforms, therefore, the summation of serum apelin-13, apelin-17, apelin-28, apelin-31, and apelin-36 was measured. The levels of TGF-β1 in supernatants of THP-1 macrophages were measured with a TGF-β1 ELISA kit (Thermo Scientific, USA). All procedures were performed according to the manufacturer's protocol. Briefly, samples were added into pre-coated ELISA plates and incubated with corresponding detection antibody and HRP-conjugate, successively. After removing unbound proteins with washing buffer, the samples were incubated with substrate solution, and then the enzyme reaction was stopped by stop solution. The absorbance was measured at 450 nm with a microplate reader (Multiskan GO, USA).

Planktonic bacteria were pelleted via centrifugation, and the culture supernatant was lyophilized in a lyophilizer (ScanVac freeze dryer). The bacterial pellet, lyophilized supernatant, and pellicle biofilm were resuspended in 300 μL of an acetic-nitric reagent and incubated for 30 min at boiling temperatures. The pellets were then washed twice with sterile water, followed by adding 67% sulfuric acid with intermittent mixings and incubated at RT for 1 h. The samples were placed on an ice bath, and 1 mL of cold anthrone reagent (Fisher Scientific) was added and mixed gently. The tubes were incubated in a boiling water bath for 15 min, after which they were placed on ice. The absorbance at 620 nm was recorded with Multiskan GO.

Total RNA was extracted from 0.5 mg of embryonic midbrain tissues and from H19-7 cells (10² to 10⁷ cells) and using the mirVana®miRNA Isolation kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions and as previously described by Kerek et al. [14 (link)]. miRNAs were isolated using a two-step procedure. In the first step, samples were disrupted in a denaturing lysis buffer and then subjected to acid-phenol/chloroform extraction. The second step consisted on purification over glass-fiber filter that immobilizes the RNA which was later eluted using RNase-free water. According to the manufacturer’s instructions, no enrichment procedure is needed while isolating miRNA for expression profiling using miRNA arrays. The concentration and purity of RNA were determined at 260/280 nm by using a nanodrop spectrophotometer (Multiskan GO).

Protein concentration was calculated using the Pierce Protein Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Each sample was tested in duplicate and absorbance was measured at 562 nm using a Multiskan Go microplate spectrophotometer.

Cells were seeded in 96 well-plates with 100 μL medium and incubated overnight before hyperthermic exposure. The next day, the medium was refreshed and cells were exposed to 42°C or 43°C for 30 minutes, or for 1 hour in a 5% CO₂ incubator. Cells maintained at 37°C were set as control. At the end of exposure, cells were returned back to 37°C for recovery. Cell viability levels were determined at 4, 24, 48, and 72 hours post exposure, by using the WST-8 cell counting kit; according to the manufacturer’s protocol (Sigma-Aldrich, Germany). This assay uses tetrazolium salt which is converted to a fluorescent product formazan by metabolically active cells. Fluorescence was monitored at 450 nm by ELISA reader Multiskan Go. Cell viability was expressed as a percentage of control cells. Five replicates (n = 5) of each experimental condition were performed.

Planktonic bacteria were pelleted via centrifugation, and the culture supernatant was lyophilized in a lyophilizer (ScanVac freeze dryer). The bacterial pellet, lyophilized supernatant, and pellicle biofilm were resuspended in 300 μL of an acetic-nitric reagent and incubated for 30 min at boiling temperatures. The pellets were then washed twice with sterile water, followed by adding 67% sulfuric acid with intermittent mixings and incubated at RT for 1 h. The samples were placed on an ice bath, and 1 mL of cold anthrone reagent (Fisher Scientific) was added and mixed gently. The tubes were incubated in a boiling water bath for 15 min, after which they were placed on ice. The absorbance at 620 nm was recorded with Multiskan GO.