Anti cd20 fitc

The cells in Teflon vials were incubated in an ice-cold water bath for 15 min, re-suspended by pipetting and fixed in 2 % PFA for 20 min at room temperature (RT). Fixed cells were washed twice by pelleting at 540 x g for 3 min and re-suspended in FACS buffer (3 % FCS, 2 mM EDTA, 0.01 % sodium azide in PBSdef). The cells were permeabilized by incubation for 30 min at RT in saponin buffer (0.5 % saponin, 3 % FCS, 2 mM EDTA, 0.01 % sodium azide in PBSdef) and washed twice with FACS buffer. Infected cells were detected by immune-staining with monoclonal antibody against viral NP protein (Abcam, ab43821) and Alexa633-labelled anti-rabbit antibodies (Invitrogen, A21052). Cell type specific surface markers were immune-stained using the following fluorescently-labelled antibodies according to manufacturer’s protocols: anti-CD3-FITC (BD Pharmingen, 555332), anti-CD4-FITC (BD Pharmingen, 555346), anti-CD8-PE (BD Bioscience, 340046), anti-CD20-FITC (Miltenyi Biotec, 130-111-337), anti-CD56-FITC (BioLegend, 318303), anti-CD14-PE (BD Pharmingen, 555399), anti-CD303-FITC (Miltenyi Biotec, 130-113-192), anti-CD1c-FITC (Miltenyi Biotec, 120-000-888). All incubations with antibodies were performed for 45 min at 4°C. Flow cytometric analysis was done using BD FACSCalibur with Cell Quest Pro V5.2 for collection and FlowJo v10.0 for analyzing of the data.