Isolation of Erythrocytes from Heparinized Blood

RBC were isolated from the heparinized blood collected from the cohabitant group in Challenge Experiment # 2 by using a previously described Percoll gradient with minor modifications [32 (link)]. In short, blood was diluted 1:10 in distilled PBS (dPBS) and a discontinuous Percoll gradient (GE Healthcare, Uppsala, Sweden) was prepared using 1.07 g/mL (49%) and 1.05 g/mL (34%). The diluted blood was layered onto the gradient and centrifuged at 2000 × g for 20 min at 4 °C. The RBC were collected from the bottom of the gradient; however a variable amount of erythrocytes was present at the 34%-49% interface. In order to collect the remaining erythrocytes, the interface was re-layered onto a fresh 34%-49% gradient and centrifuged at 500 × g for 10 min at 4 °C. The RBC from the bottom of the gradient were collected, pooled with the erythrocytes from the first gradient, and washed twice in PBS. The cells were counted using Countess (Invitrogen, Eugene, Oregon, USA) and resuspended in PBS to a final concentration of 5 × 10⁶ cells/mL. Smears of the isolated RBC stained with Diff-Quik (Dade Behring, Newark, USA) confirmed that the isolated cells were erythrocytes.