Ammonium chloride potassium ack lysing buffer

Blood from interstate blood bank Inc. (Memphis, TN) was diluted with phosphate-buffered saline (PBS, Life Technologies, NY), layered on Ficoll (Mediatech Inc. Manassas, VA) and centrifuged at 400g for 30 minutes. The white ring of peripheral blood mononuclear cells (PBMCs) formed in between the plasma and Ficoll layers were carefully isolated. The PBMCs were washed with PBS several times to ensure the removal of Ficoll. The cells were incubated with ammonium-chloride-potassium (ACK) lysing buffer (Life Technologies, NY) at 4°C for 15 minutes to lyse and remove any red blood cells, if present. The clear pellets of PBMCs were then cultured in RPMI media with human serum and macrophage colony-stimulating factor for macrophage differentiation. The differentiated macrophages were treated with BaP (100 nM) for 6 days, every 24 hours with a addition of 500 μl fresh media after each treatment.

Neutrophils were isolated from whole blood in CPTTM tubes as previously described (Heit et al., 2006 (link); Lindblom et al., 2018 (link)). Briefly, whole blood was subjected first to dextran sedimentation using 6% Dextran/0.9% NaCl solution at room temperature for 45-60 min. Supernatant containing neutrophils and mononuclear cells was transferred to a conical tube and centrifuged for 12 min at 300 RCF, 4°C with low brake. Neutrophils and mononuclear cells were separated from the erythrocyte-rich pellet by lysing erythrocytes with Ammonium-Chloride-Potassium (ACK) Lysing buffer (ThermoFisher). The pellet was resuspended in phosphate buffered saline (PBS) and the cell suspension was layered over Ficoll-Histopaque-1077 (Catalog No.1077, Sigma) and centrifuged for 30 min at 450 RCF, room temperature, low brake. After centrifugation, PBMCs were removed, and the pellet that contained neutrophils was further processed. Neutrophils were rinsed with 4 mL of Hanks’ Balance Salt Solution (HBSS), centrifuged at 450 RCF for 5 min and resuspended in 2 mL HBSS. Cell count and viability data were obtained using the MUSE cell analyzer system (Millipore). Cells were cryopreserved using 30% dimethyl sulfoxide (DMSO) in RPMI medium and thawed for later use following a previously reported protocol (Milani et al., 2016 (link)). A flowchart of the methods is shown in the Supplementary Figure S1.

Prior to myringotomy, the ears were rinsed from cerumen and flushed with 1 ml sterile saline. This external ear canal (EEC) lavage fluid was collected for microbiome analyses. The ear canals were subsequently washed once with 70% denatured alcohol. After incision of the tympanic membrane, the middle ear effusion (MEE) was aspirated into a sterile trap, and 1 ml sterile saline was used to clear the suction catheter. Nasopharyngeal (NPH) swabs were collected and swirled in 1 ml sterile saline. See Fig. S1B for a summary of sample collection procedure. Besides use for the analyses in this study, all MEE and NPH samples were also routinely sent to the Department of Clinical Microbiology, Skåne University Hospital, for identification of bacteria.
Aliquots of all samples were immediately frozen at −80°C for subsequent microbiome analyses. Cells were collected by centrifugation at 350 × g for 10 min. The supernatants were collected and stored at −80°C until used for analyses of inflammatory mediators. Red blood cells in the cell pellets were lysed using ammonium-chloride-potassium (ACK) lysing buffer (Thermo Fisher Scientific). The cells were washed, and the total number of cells in the samples, as well as the percentage of viable cells, was calculated by the trypan blue exclusion assay.

SVF cells were isolated from healthy female Sprague-Dawley rats as described elsewhere [9 (link)]. In summary, fat was minced in 0.2% collagenase type I (Thermo Fisher Scientific; #17100017) in HBSS and placed in a 37 °C water bath for 60 min. The enzyme solution was neutralized using Minimum Essential Medium (MEM; Thermo Fisher Scientific; #32561029) supplemented with 10% FBS and filtered through a 70-μm cell strainer. Then, red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) lysing buffer (Thermo Fisher Scientific; #A1049201) for 3 min. ACK buffer was removed by washing/centrifugation and the resulting SVF pellet was resuspended in MEM supplemented with 10% FBS and 1% penicillin-streptomycin.

For the tumor killing assay, OT-1 cells were cultured 1:10 with RCT-OVA-4-1BBL, RCT-OVA-4-1BBL-IL-7, RCT-OVA-4-1BBL-IL-12, or RCT-OVA-4-1BBL-IL-15 for 3 days. OT-1 cells were then harvested and treated with an Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Thermo Fisher Scientific). Approximately 1 × 10⁴ CellTrace Far Red dye-labeled (Thermo Fisher Scientific) EL4 (control target) or EG7.OVA (target) cells were then incubated with expanded OT-1 cells (effector cells) at a 5:1, 2:1, 1:1, 0.5:1, or 0:1 effector-to-target ratio. After a 22-h incubation period, cells were stained with LIVE/DEAD^™ Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific) and fixed with 2% paraformaldehyde (Thermo Fisher Scientific) to enumerate live target cells (CellTrace Far Red dye⁺) in each well.