Lungs and spleens were processed as described by Fox et al. [25 (link)]. Briefly, a portion of the left cranial lung lobe and spleen from each hamster were aseptically transferred from PBS into DMEM then teased apart. Lungs were treated with a solution of DNase IV (500 units/mL) and Liberase (0.5 mg/mL) for 30 min at 37 °C to dissociate and digest collagen. Both lung and spleen cells were homogenized using a syringe plunger and passed through a 70 µm filter to prepare single cell suspension. Erythrocytes were lysed using Gey’s RBC lysis buffer (0.15 M NH4Cl, 10 mM HCO3) and cells were resuspended in 1 mL of complete media.
For blood, buffy coat was harvested by adding equal volume of PBS + 2% FBS to the blood and centrifuging at 800× g for 10 min at 25 °C with brakes off. The buffy coat was collected and washed, and erythrocytes were lysed using 1× Miltenyi RBC lysis buffer (Miltenyi, CA, USA). Cells were washed and resuspended in 1 mL complete media. After adding absolute counting beads (Invitrogen), total cell numbers of lung, spleen and blood were determined by flow cytometry analysis using an LSR-II (BD).
For blood, buffy coat was harvested by adding equal volume of PBS + 2% FBS to the blood and centrifuging at 800× g for 10 min at 25 °C with brakes off. The buffy coat was collected and washed, and erythrocytes were lysed using 1× Miltenyi RBC lysis buffer (Miltenyi, CA, USA). Cells were washed and resuspended in 1 mL complete media. After adding absolute counting beads (Invitrogen), total cell numbers of lung, spleen and blood were determined by flow cytometry analysis using an LSR-II (BD).
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