Isolation of Immune Cells from Mouse Spleen and Lymph Nodes

Mice were euthanized by CO₂ asphyxiation followed by cervical dislocation and immersed in 75% ethanol for 3 to 5 minutes, then transferred to the biosafety cabinet. The spleen and cervical lymph nodes (CLN) were collected and placed in sterile 6-well plates (Corning Costar, cat. #07-200-83) with 3 mL 1× phosphate-buffered saline (PBS; ThermoFisher, cat. #10010) on ice. Spleen tissues were minced with a razor (ThermoFisher, cat. #G535010) and CLNs were dissociated with two frosted slides (ThermoFisher, cat. #12-548) followed by digestion with 1× PBS solution including 1 mg/mL collagenase A (Worthington, cat. #LS004174), 1 mg/mL DNase (MilliporeSigma, cat. #10104159001), and 2% heat-inactivated FBS (R&D Systems, cat. #S11550H) for 20 minutes at 37°C with 5% CO₂. The digested tissues were then passed through 70-mm cell strainers (MilliporeSigma, cat. #CLS431750) using mechanical force with the rubber end of a 5-mL syringe. Cell suspensions were then treated with 0.1M EDTA (ThermoFisher, cat. #15575) for 5 minutes at 37°C and washed 2 times with 1×PBS. After lysis of red blood cells with ammonium chloride potassium (ThermoFisher, cat. #A1049201), cells were collected by centrifugation at 450 × g for 10 minutes at 4°C.