Stomacher 80 biomaster blender

Splenocytes were prepared by aseptically removing the spleens from naive C57BL/6 mice. Spleens were homogenized into single-cell suspensions using a Stomacher 80 Biomaster blender (Seward, Davie, FL). The resulting suspension was centrifuged at 1600×g for 30 min and then subjected to red blood cell lysis (Sigma-Aldrich) according to the manufacturer’s instructions. The single splenocytes were collected and plated in a 96-well plate in RPMI 1640 media supplemented with heat inactivated 10% fetal bovine serum, 10 mM l-glutamine, 10 mM HEPES, 50 µM β-mercaptoethanol, and 100 µg/ml penicillin/streptomycin at a density of 1 × 10⁶ cells/well at 37 °C. Splenocytes were stimulated with PBS or 1 µg/ml SEB, followed by treatment with BBR (1, 2, 4, or 8 μM) or 100 nM tichostatin A (TSA) for 24 h. To assess activation, splenocytes collected from in vitro culture were stained with anti-mouse CD69 antibody (Biolegend, San Diego, CA, USA). Flow cytometry analysis was conducted in splenocytes using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the number of splenocytes was analyzed by Cell Quest Pro (BD Biosciences).

These cells were isolated as described previously (Rieder et al., 2012 (link)). Briefly, mice were given heparinized PBS to perfuse the lungs. A stomacher 80 biomaster blender (Seward, Fl) was used to homogenize the lung tissue suspended in 10 ml of 1× PBS and 10% Fetal Bovine Serum (FBS). The homogenized lung tissues were centrifuged at 1,200 rpm at 4°C for 12 min. The pellet was then washed with PBS and resuspended in PBS post centrifugation. We then used a density gradient centrifugation method to purify lung mononuclear cells (MNCs). In brief, the cells resuspended in sterile PBS were carefully layered using Histopaque-1077 purchased from Sigma-Aldrich (St. Louis, MO) and centrifuged at 500 × g for 30 min at room temperature (25°C). The mononuclear cells were collected at the interface. MNCs mixed with trypan blue were then enumerated using a Biorad TC 20-Automated cell counter.