Murine igg

The IgG-dexamethasone conjugate was synthesized by conjugating dexamethasone (on average, four drug molecules per IgG) to primary amino groups of either the murine anti-rat CD163 antibody (ED2; Bio-Rad AbD Serotec) or to murine IgG (Sigma-Aldrich) as described previously using a hemisuccinate linker.²⁷ (link)

Cells were harvested by washing once with phosphate-buffered saline (PBS) and treating with 1× trypsin/EDTA for 1 min at 37°C. After neutralizing the trypsin with growth medium, the cells were washed once in flow cytometry (FC) buffer (1% bovine serum albumin and 0.05% sodium azide in PBS). The cells were resuspended in an unconjugated primary antibody (murine IgG, Sigma Aldrich, Poole, UK, 1∶1000 dilution in FC buffer; anti-MICA clone AM01, anti-MICB clone BM02, anti-MICA/B clone BAM01, anti-ULBP2 clone BUM01, BAMOMAB GmBH, Graefelfing, Germany, 1∶400 dilutions in FC buffer; anti-ULBP1 Clone 170818, MAB1380, anti-ULBP3 Clone 166510, MAB1517, R&D Systems, Abingdon, UK, 1∶200 dilutions in FC buffer; anti-MHC-I, clone W632, AbD Serotec, Kidlington, UK, 1∶2000 dilution in FC buffer) and incubated for 30 min at 4°C. The cells were then washed twice with FC buffer and incubated in an Alexa Fluor 647 conjugated anti-mouse IgG secondary antibody (Invitrogen, 1∶500 dilution in FC buffer) for 30 min at 4°C. After 3 further washes in FC buffer, the cells were fixed in 2% paraformaldehyde (PFA) for 10 min and analyzed by using an Accuri Cflow cytometer (BD Biosciences, Oxford, UK). A forward scatter and side scatter dot plot was used to gate both on viable cells and infected cells (Fig. S1A). The median fluorescence intensity (MFI) was used in subsequent analysis.