Ebioscience rbc lysis buffer

Anaesthetized mice were perfused with 1X HBSS (10U/ml heparin) and forebrains, spinal cord and spleens were collected in ice-cold 1X HBSS. Brains were dissociated using the Papain Dissociation System (Worthington Biochemical) by gentle sequential trituration, before and after incubation of 30 min at 37°C and 5% CO₂. Single cell suspensions were filtered through a 40 µm cell strainer (Falcon) and resuspended in 30% Percoll^® (Sigma-Aldrich) in AutoMACS running buffer at room temperature and centrifuged for 15 min at 500 xg at room temperature with no brake to allow for formation of a floating myelin layer. Following myelin removal, pelleted cells were resuspended in AutoMACS running buffer (Miltenyi Biotec), filtered again, and centrifuged at 300 xg for 10 min to obtain an immune cell-enriched pellet. Spleens were homogenized by mechanical dissociation followed by red blood cell removal using 1X eBioscience RBC lysis buffer (ThermoFisher) for 10 min at room temperature and filtered through a 40 µm cell strainer.

The oxidative burst of blood neutrophils was evaluated in rats after 5 weeks of dLAN. In this procedure, 50 µl of whole blood was diluted 20 times with PBS containing 20 µM of 2′,7′-dichlorodihydrofluorescein diacetate (H₂-DCF-DA; MilliporeSigma, USA; D6883) and incubated for 30 min at 37°C in the dark. Thereafter, each sample was divided into two equal parts; one part was stimulated with phorbol-12-myristate-13-acetate (PMA; MilliporeSigma; P8139) at a final concentration of 1 µM and one part served as the unstimulated control (dimethyl sulfoxide was added at a final concentration of 0.1%). After incubation (for 30 min at 37°C in the dark), the cells were washed with PBS and red blood cells were lysed using eBioscience™ RBC Lysis Buffer (Thermo Fisher Scientific; 00-4333-57). After lysis of erythrocytes, the cells were washed, resuspended in 0.3 ml of PBS, and analyzed on a flow cytometer. Within the cell, H₂-DCF-DA is converted to fluorescent 2′,7′-dichlorofluorescein (DCF), and this fluorescence is a measure of H₂O₂ production and is linearly related to the oxidative burst of the stimulated neutrophils (28 (link)). The functional activity of the neutrophils was expressed as a fold increase in the median DCF fluorescence of the PMA stimulated samples over the unstimulated samples.

Spleens from mice were collected and processed into single-cell suspensions in RPMI1640 media supplemented with 10% heat-inactivated fetal calf serum and penicillin/streptomycin (R10 media). Red blood cells were lysed using eBioscience RBC lysis buffer (Thermo Fisher Scientific) and resuspended in R10 media to stop the lysis. Splenocytes were counted and 100,000 cells per well were added to the plates of the Mouse IFN-γ ELISpot^PLUS (HRP) kit (Mabtech). Cells were then stimulated for 18 hours at 37°C with SARS-CoV-2 S PepTivator peptides (Miltenyi Biotec) or media alone as a negative control. Spots were developed as per the manufacturer’s instructions. Spots were quantified by eye and plotted as SFU per million cells.