Fc receptor binding inhibitor polyclonal antibody

FOLR1⁺ Hela cells, FOLR1⁻ LNCaP cells, and FOLR1⁻ PC3 cells grown in 10 cm culture dish were harvested (cell viability > 90%) and washed three times in ice-cold DPBS buffer supplemented with 0.5% BSA and aliquoted to a density of 5 × 10⁶ cells/mL in flow cytometry staining buffer (FC001, R&D Systems, Minneapolis, MN, USA). Next, flow cytometric analyses were performed following standard protocols. In brief, 5 × 10⁵/100uL cells were incubated with Fc Receptor Binding Inhibitor Polyclonal Antibody (14-9161-73, eBioscience, San Diego, CA, USA) on ice for 20 min. Without washing, staining proceeded with primary antibody PE anti-FOLR1 antibody (908303, BioLegend) incubation followed by flow cytometric analysis using LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software v10 (Tree Star Inc., Ashland, OR, USA). For the functionality of FOLR1, Hela cells, LNCaP cells, and PC3 cells were incubated with folate–NIR (50 nM) for the indicated time periods followed by flow cytometric analyses as described above.

To assay maternal cells’ presence in the pups’ lymphoid organs, cell suspensions were prepared from postnatal day 13-15 pups’ spleen and thymus by mechanical dissociation using 70 μm nylon mesh (Axel). Cell suspensions were treated with red blood cells lysis buffer (ammonium-chloride-potassium) and washed in staining buffer [HBSS(-), 2 mM EDTA, 1% BSA]. Single cells were stained in a 96-well plate at a concentration of 3-5×10⁶ cells per well. Cells were stained simultaneously with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (eBioscience) at 1 μl/well, H2Kb-FITC (BioLegend clone AF6-88.5) and H2Dd-PE (BioLegend clone 34-2-12) at a concentration of 1/75th for 30 min at 4°C; fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences); blocked with Fc Receptor Binding Inhibitor Polyclonal Antibody (eBioscience) at 20 μl/well for 15 min and stained intracellularly with HB-EGF-APC (R&D Systems clone 125923) at a concentration of 1/10th for 30 min at room temperature. The following day, 20-30×10⁶ cells per sample were analyzed and sorted on a fluorescence-activated cell sorter (BD FACSAria III) using the BD FACSDiva software (Becton-Dickinson). Sorting was performed by gating for live singlets and maternal cells’ markers on gates H2Kb⁺H2Dd^-Hbegf⁺ and H2Kb^intH2Dd⁺Hbegf⁺ for F1 and F2 pups, respectively, and the ratio of sorted cells over live cells R1 was recorded.

hPMN were centrifuged at 300 g for 5 min, washed with PBS, and incubated for 15 min with Zombie near IR fixable viability dye followed by 15-min incubation with an Fc receptor binding inhibitor polyclonal antibody (eBioscience). Cells were washed in PBS containing 1% bovine serum albumin. Cells were stained with a human A2aR Alexa Fluor 488-conjugated antibody (Clone #599717) or CD39 PE-conjugated antibody [eBioA1 (A1)] for 15 min and fixed with Fix & Perm fix A (Thermofisher) for 15 min. Samples were assessed using a BD FACSCanto II (BD biosciences) cytometer.
To measure ROS, hPMN were infected as above, and at 2.5 h post-gentamicin treatment, they were incubated with the cell-permeable ROS sensor CellRox green reagent for 30 min and treated as above.