Gentle macs tubes c

Spleens, lungs, inguinal lymph nodes were aseptically harvested from euthanized mice and processed to extract single cell suspensions in accordance with established methodology^{33 (link)}. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec), followed by 1 h collagenase digestion (Sigma Aldrich; C5138) at 37 °C, 5% CO₂. The lung homogenate, spleens, and lymph nodes were subsequently forced through 70-µm cell strainers (BD) with the plunger from a 3 mL syringe (BD). Cells were washed twice in cold RPMI or PBS followed by 5 min centrifugation at 700 ×g. Finally, cells were resuspended in supplemented RPMI media containing 10% fetal calf serum (FCS). Cells were counted using an automatic Nucleocounter (Chemotec) and cell suspensions were adjusted to 2 × 10⁵ cells/well for ELISA and 1–2 × 10⁶ cells/well for flow cytometry.

Spleens or lungs were aseptically harvested from euthanized mice. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec) or chopped into small pieces using scalpels, followed by 1 h of collagenase digestion (Sigma-Aldrich; catalog no. C5138) at 37°C and 5% CO₂. The lung homogenate and spleens were forced through 70- to 100-μm cell strainers (BD Biosciences) with the stopper from a 5-ml syringe (BD) and washed twice with cold RPMI medium (Gibco; RPMI 1640) by centrifuging 5 min at 1,800 rpm. A red blood cell lysis step was performed in between washes (Roche, catalog no. 11814389001). Cells were finally resuspended in enriched RPMI medium (RPMI 1640, 10% heat-inactivated fetal calf serum (FCS) (Biochrom GmbH), 10 mM HEPES (Invitrogen), 2 mM l-glutamine (Invitrogen), 1 mM Natriumpyruvate (Invitrogen), 1× nonessential amino acids (MP Biomedicals, LLC), 5 × 10⁵ M 2-mercaptoethanol (Sigma-Aldrich), and penicillin-streptomycin (Gibco). Cells were counted using an automatic Nucleocounter (Chemotec) and adjusted to 2 × 10⁵ cells/well for enzyme-linked immunosorbent assay (ELISA) and 1  × 10⁶ to 2 × 10⁶ cells/well for flow cytometry.

Spleens or lung were aseptically harvested from euthanized mice. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec) followed by 1 h of collagenase-digestion (Sigma Aldrich; C5138) at 37 degrees, 5% CO₂. The lung homogenate and spleens were forced through 100 µm cell strainers (BD Biosciences) with the stopper from a 5 ml syringe (BD) and washed twice with cold RPMI medium (Gibco; RPMI-1640) by centrifuging 5 min at 1,800 rpm. Cells were finally re-suspended in enriched RPMI medium [RPMI-1640, 10% heat-inactivated FCS (Biochrom Gmbh), 10 mM Hepes (Invitrogen), 2 mM L-Glutamine (Invitrogen), 1 mM Natriumpyruvate (Invitrogen), 1× Non-essential amino acids (MP Biomedicals, LLC), 5×10^-5 M 2-mercaptoethanol (Sigma-Aldrich), and PenStrep (Gibco)]. Cells were counted using an automatic Nucleocounter™ (Chemotec) and adjusted to 2x10⁵ cells/well for ELISA and 1-2x10⁶ cells/well for flow cytometry.