Red blood cell lysing buffer hybri max

Bone marrow from LDLr^−/− male mouse femurs was flushed using an L929 conditioned medium as mentioned above. The cellular suspension was centrifuged at 1,000 × g at room temperature, and the supernatant was discarded. Pelleted cells were incubated with 1.0 ml Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich, R7757, St. Louis, MO) for 3 min at room temperature to lyse red blood cells. Lyses were stopped using 1.0 ml of Dulbecco’s Phosphate Buffered Saline solution (Sigma-Aldrich, D8537). Cells were centrifuged at 1,000 × g at room temperature, and the supernatant was discarded. Then, the bone marrow cells were resuspended in a differentiation medium: 69% (v/v) DMEM, 20% (v/v) L929 conditioned medium, 1% (v/v) Penicillin–Streptomycin Mixture (Lonza, 17-602F, Basel, CH), and 10% (v/v) fetal bovine serum (Vitrocell, 11). Cells were plated in a 100 mm diameter Petri Dish (Nest Biotechnology, 753001, Wuxi, China) and incubated at 37°C and 5% (v/v) CO₂ for 7 days for full differentiation, changing half part of the differentiation medium at day 3. Bone marrow-derived macrophages (BMDMs) were carefully detached using 1.5 ml Accutase Cell Detachment solution (Sigma-Aldrich, SCR005), counted in a hemocytometer, and plated in a 24-well plate (Nest Biotechnology, 702001) or 96-well plate (Greiner Bio-one, 655090, Kremsmünster, AT) for further analysis.

PBMCs were isolated as described previously [11 (link)]. Briefly, blood samples were collected into lithium-heparin coated tubes and diluted with equal volumes of phosphate buffered saline (PBS). The diluted mixture was layered over a half of the volume of Lymphoprep (Axis-shield, Oslo, Norway) and was separated by gradient centrifugation at 800 × g for 30 min at RT. Contaminating red blood cells were lysed using commercial Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich, St. Louis, MO, USA). The PBMCs were then washed twice with PBS prior to resuspension in a complete medium consisting of RPMI-1640 medium (Lonza, Basel, Switzerland) containing 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 2% (v/v) of the antibiotic-antimycotics formulating solution with penicillin, streptomycin, and amphotericin B (Lonza, Basel, Switzerland).

Lungs were isolated, finely minced, and digested in Roswell Park Memorial Institute (RPMI)-1640 containing 5% fetal bovine serum (FBS), 0.2 mg/ml of Liberase™ TM (Roche), and 0.1 mg/ml of DNase I (Roche) at 37°C. Digested lungs were homogenized with a 5-ml syringe attached to an 18G needle, filtered through a 70 μM cell strainer, and washed with PBS. Red blood cells were lysed using Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma-Aldrich), and cells were washed with fluorescence-activated cell sorting (FACS) buffer (PBS + 2% FBS). Small intestines were isolated, opened longitudinally, cut in small pieces, and vortexed in gut buffer (Hanks’ Balanced Salt Solution (HBSS) + 2% FBS + 15 mM of HEPES) to remove fecal matter. Intestinal epithelial cells were removed by incubation in gut buffer containing 5 mM of EDTA for 20 min. Lamina propria was subsequently digested in RPMI-1640 supplemented with 5% FBS, 15 mM of HEPES, 0.1 mg/ml of Liberase™ TM (Roche), and 0.1 mg/ml of DNase I (Roche) at 37°C for 15 min. Digestion suspensions were filtered through a 70 μM cell strainer to obtain a single-cell suspension, and cells were washed with FACS buffer.