Facsdiva software vision 8

Before euthanasia through decapitation, mice were sedated using inhalation anesthesia (isoflurane 2.5% at 1.5 L/min in room air). Whole blood was collected and the trachea cannulated. Spleen was extracted before trans-cardiac perfusion, whereafter the lung-heart block was extracted, and a broncho-alveolar lavage was performed by instilling 1 mL of sterile PBS. For flow cytometry experiments, the left lung was cut into pieces and enzymatically digested in a DNAse-collagenase XI mix under continuous agitation at 37 °C before the homogenate was passed through a 40 µm cell strainer. After centrifugation, red blood cells were lysed, and the cell pellets were incubated in Fc block prior to staining with antibodies (see Appendix A, Table A1). As for the spleen, red blood cells were lysed after homogenization of the tissue through a 40 µm cell strainer. Cell pellets were incubated in Fc block prior to staining with antibodies (see Appendix A, Table A1). Data acquisition was carried out in a BD LSR Fortessa cytometer using FacsDiva software Vision 8.0 (BD Biosciences). Data analysis was performed with FlowJo software (version 10, TreeStar Inc., Ashland, OR, USA). Cells were plotted on forward versus side scatter and single cells were gated on FSC-A versus FSC-H linearity.

Whole blood from mice was collected in EDTA-coated tubes and red blood cells were lysed before samples were incubated in F_c block solution followed by primary antibodies. After washing and centrifugation, the supernatant was decanted, and pellets were re-suspended in FACS buffer. Brain tissue was enzymatically digested and homogenized. After density separation using Percoll (GE Healthcare), pellets were reconstituted in F_c block prior to staining with antibodies (Supplementary Table 5). Data acquisition was carried out in a BD LSR Fortessa cytometer using FacsDiva software Vision 8.0 (BD Biosciences). Data analysis was performed with FlowJo software (version 10, TreeStar Inc., USA). Cells were plotted on forward versus side scatter and single cells were gated on FSC-A versus FSC-H linearity.