Fitc conjugated anti b220

Peritoneal cells were harvested from BALB/c mice. Analyses of apoptosis were performed as described elsewhere (34 (link)). The cells were then plated in 24-well plates at a density of 1 × 10⁶ cells per well. If necessary, SNA (10 μg/ml) pretreatment was applied. A/WSN/1933 virus or UV-WSN virus was added to each well at a 1 × 10⁶ pfu. After incubation at 37°C for different time periods, cells were harvested and stained with anti-FcγRII/III (Fc receptor blocker) before being stained with APCef780-conjugated anti-CD19 (Catalog No: 47-0193-80, eBioscience), PE-conjugated anti-CD23 (Catalog No: 12-0232-81, eBioscience), FITC-conjugated anti-B220 (Catalog No: 553088, BD Biosciences), FITC-conjugated anti-F4/80 (eBioscience), and BV421-conjugated anti-CD11b antibodies (Catalog No: 48-0112-80; eBioscience). After 1 h incubation at 4 °C, cells were washed with FACS buffer and stained with APC-conjugated Annexin V (Catalog No: 17-8007-74, eBioscience) for 15 min at room temperature. To exclude dead cells, stained cells were incubated with 10 μg/mL of PI (eBioscience, San Diego, CA, USA) or 7AAD (Catalog No: 51-68981E, BD Biosciences). The cells were analyzed with a FACSCanto^TM II.

Purified hcAbs were conjugated via amino groups to Alexa Fluor⁶⁴⁷-fluorochrome according to the manufacturer’s instructions (Molecular Probes, Thermo Fisher Scientific). For epitope mapping analyses, EL4 cells were pre-incubated with a saturating concentration (100 nM) of unconjugated hcAbs for 30 min at 4°C, followed by addition of Alexa Fluor⁶⁴⁷-conjugated hcAbs (10 nM) and further incubation for 20 min at 4°C. Cells were washed and analyzed by flow cytometry on a BD-FACS Canto. Data was analyzed using the FlowJo software (Treestar). The percentage of cross-blockade was calculated from mean fluorescence intensities (MFI) as follows: (MFI in the absence of competing Abs – MFI in the presence of competing Abs): (MFI in the presence of competing Abs) x 100. Spleen cells were pre-incubated with Fc-block (BioXcell, clone 2.4G2) to minimize unspecific binding to Fc-receptors. Cells were then incubated with Alexa Fluor⁶⁴⁷-conjugated hcAbs, FITC-conjugated anti-B220 (BD biosciences, clone RA3-6B2), and Alexa Fluor 750 as a viability dye (ThermoFischer). Gating was performed on Alexa Fluor 750-low cells (live cells).

Mice were sacrificed 20 hours after induction of CASP by cervival dislocation under deep anaesthesia. Spleens were taken and splenocytes were isolated. After blocking with FcBlock (BD Biosciences, San Jose, California, USA) cells were stained with appropriate antibodies according to the manufacturer’s instructions. PE-conjugated anti-mouse DR5 and PE-conjugated anti-mouse TRAIL were purchased from eBioscience. Isotype controls (PE-conjugated Armenian Hamster IgG and Rat IgG2ak) were also purchased from eBioscience. APC- and FITC-conjugated anti-Ly6G (clone 1A8) was purchased from Miltenyi (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Rat IgG2a (Miltenyi) was used for isotype control. FITC-conjugated anti-B220, FITC-conjugated anti-CD3, PE-conjugated anti-CD11b and FITC-conjugated anti-Ly6C was purchased from Pharmingen (BD Biosciences, San Jose, California, USA). Appropriate isotype controls were also purchased from Pharmingen.
FACS analysis was done using a BD FACS Calibur system. For cell analysis FlowJo (Treestar Inc., Ashland, USA) and WinMDI software were used.