Red cell lysis buffer

Mice were sacrificed eight days post prime or prime-boost immunizations. A cell suspension was prepared by homogenizing the spleens through 200 µm mesh sieves and red blood cells were removed by adding Red Cell lysis buffer (Sigma, St. Louis, MO, USA). After centrifugation, the cell pellet was resolved in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine and 100 IU/mL penicillin/streptomycin. Interferon-γ secreting CD8+ T cells were analyzed by ELISPOT assay (ELISPOTPLUS Kit for mouse IFN-γ, MABTECH, Eching, Germany) following the manufacturer’s instructions. ELISPOT plates were pre-incubated overnight with the antibody solution and then incubated with the cell suspension that had been stimulated with the EBOV-specific peptides NP_44–52 (YQVNNLEEI; [45 (link)]) and NP_388–396 (FQQTNAMVT; [45 (link)]). The spots were counted and analyzed by using an automated ELISPOT plate reader and software following the manufacturer’s instructions (A.EL.VIS Eli.Scan, A.EL.VIS ELISPOT Analysis Software, Hannover, Germany).

Lymphocytes from spleens (SPL), mesenteric lymph nodes (MLN), and Payer’s patches (PP) were isolated according to standard protocols used in our laboratory [24 (link)]. Briefly, tissues were homogenized in RPMI 1640 medium supplemented with 10 mM HEPES and 10 units/mL penicillin-streptomycin solution (incomplete medium), and the resulting cell suspension was filtered through an 80-μm nylon filter. The splenocytes were incubated with red cell lysis buffer (Sigma) for 5 min to remove erythrocytes. The lymphocytes were washed with incomplete medium and centrifuged at 400× g at 10 °C for 10 min. The pelleted cells were resuspended in 1 mL of incomplete medium. After trypan blue (Sigma) staining, lymphocytes were counted using a Bruker cell counter.

Lymphoid tissue was dissociated using standard protocols and red blood cells removed using Red Cell Lysis buffer (Sigma). Unless otherwise stated, cells were cultured in complete RPMI 1640 (Lonza; supplemented with 25 mM HEPES, 50 U/ml Pen/Strep, 2 mM L-Glutamine and 50 µM 2-mercaptoethanol) with 5–10% FCS (BioSera, Hyclone). PL8 and CH27 cells were maintained in complete RPMI with 10% FCS. Th17 cells were generated and maintained in IMDM (Lonza; supplemented with 50 U/ml Pen/Strep, 2 mM L-Glutamine and 50 µM 2-mercaptoethanol) containing 10% FCS.

Peripheral blood mononuclear cells (PBMCs) from healthy anonymous donors were isolated from leukocyte provided by the National Blood and Transplant Service, UK. Red blood cells were removed by centrifugation on Ficoll-Paque (28-4039-56 AD; GE Healthcare) and red cell lysis buffer (11814389001; Sigma-Aldrich). Recovered PBMCs were washed to remove platelets. Monocytes were isolated from PBMCs using a magnetic cell separation system with anti-CD14 mAb-coated microbeads (130-050-201; Miltenyi Biotec). CD14-positive monocytes were cultured in complete RPMI 1640 medium (Lonza) supplemented with 9.1% heat-inactivated FCS supplemented with either 50 ng/ml GM-CSF or 50 ng/ml M-CSF (130-093-862/130-093-963; Miltenyi Biotec) at 37°C under a humidified 5% CO₂ atmosphere for 6 d. Media were replaced at 3 d. On day 6, the cells were washed and detached with 0.5 mM EDTA in ice-cold PBS and plated in a 24-well plate (containing coverslips for microscopy) or a live-cell imaging dish at a concentration of 2 × 10⁵ cells/well with complete RPMI media (for resting macrophages). Macrophages were either treated overnight or after infection with recombinant human IFN-γ (PHC 4031; Gibco) at a concentration of 100 U/ml.

Splenocyte suspensions were obtained using a glass homogeniser (Fisher) with RPMI containing 10% FBS, 100 IU/ml penicillin and 0.1 mg/ml streptomycin (Gibco). Cells were treated with red cell lysis buffer as directed (Sigma-Aldrich). Live cells were counted using Trypan blue exclusion.