Bio plex pro mouse cytokine 23 plex immunoassay kit

Mice were euthanized with ketamine/xylazine, and lungs were lavaged with total 1.8 mL saline (0.6 mL/each time, for a total of 3 times). The BAL fluids were spun down to collect inflammatory cells, and supernatants were transferred for cytokine analysis. The cells were suspended and labeled with specific antibodies to identify the differentiated inflammatory cells. Specific cells were identified by Guava easyCyte flow cytometer (Millipore Sigma) and analyzed by Guava InCyte (50 (link)). Inflammatory cytokines were measured by Bio-plex Pro mouse cytokine 23-plex immunoassay kit (M60009RDPD; Bio-Rad), and plates were read through the Luminex Flexmap 3D (Luminex Corp).

To measure the pro-inflammatory cytokines present in BALF, a Bio-Plex Pro mouse cytokine 23-plex immunoassay kit (Cat#: M60009RDPD, BioRad) was used, according to the manufacturer’s instructions. Briefly, the diluted magnetic beads were placed into the assay plate and rinsed with wash buffer. The BALF samples and standards were then added into the wells, and shaken at 850 rpm at room temperature for 30 min. After sample incubation, the plates were washed with wash buffer 3 times, then the detection antibody was added and the plates incubated for 30 min at room temperature, shaking at 850 rpm. Next, plates were washed with wash buffer 3 times, and SA-PE was added for 10 min at room temperature, shaking at 850 rpm. After this step, the plates were washed 3 times with wash buffer, and the beads were resuspended in assay buffer for reading. Results are determined via the Luminex flexmap 3d (Luminex Corp.)

The levels of pro‐inflammatory mediators in BALF from mice exposed to inhaled e‐cig aerosols containing PG and PG with nicotine and mice exposed to air (control) were analyzed by quantitative cytokine assays using the Bio‐Plex Pro mouse cytokine 23‐plex immunoassay kit (Bio‐Rad Laboratory) according to the manufacturer's instructions.