Rat igg isotype control

Cell suspensions of the dissected iLNs from the recipient mice were obtained by pressing the tissues through a 70 μm mesh in PBS with 2% FCS. Cell numbers and viability were determined using a CASY TT Cell Counter (Roche). Cells were washed and transferred into 96-well v-bottom plates. Surface antibody staining was performed for 2 hours at 4 °C in PBS with 2% FCS, in the presence of 2.4G2 hybridoma (ATCC hb-197) tissue culture supernatant and Rat IgG isotype control (Invitrogen, #10700) to block non-specific binding via Fc interactions. Following incubation, samples were washed twice with PBS with 2% FCS and acquired on an Aurora Spectral Cytometer (Cytek). Cells for single colour controls were prepared in the same manner as the fully stained samples. Flow cytometry data were analysed using FlowJo v10 software (Tree Star). The antibodies used are listed in Table 2.

A small aliquot (1-2 x 10⁶) of enriched B cells from the young adult or aged donor mice was stained for phenotypic analysis prior to transfer. Briefly, cells were stained in 96-well v-bottom plates. Surface antibody staining was performed for 2 hours at 4 °C in PBS with 2% FCS, in the presence of 2.4G2 hybridoma (ATCC hb-197) tissue culture supernatant and Rat IgG isotype control (Invitrogen, #10700) to block non-specific binding via Fc interactions. Following incubation, samples were washed twice with PBS with 2% FCS, before they were fixed with the eBioscience Foxp3/Transcription Factor Staining Buffer (#00-5323-00) for 30 min at 4°C. The samples were then washed twice with 1 x permeabilisation buffer (eBioscience #00-8333-56) and stained with the intracellular antibody mix in permeabilisation buffer at 4°C overnight. Following overnight incubation, the samples were washed twice with 1 x permeabilisation buffer and washed once with PBS with 2% FCS, before they were acquired on an Aurora Spectral Cytometer (Cytek). Cells for single colour controls were prepared in the same manner as the fully stained samples. The antibodies used for the surface and intracellular staining are listed in Table 1.

For quantification of surface receptor expression, cells were trypsinized and brought to single-cell suspension in FACS buffer (1× PBS + 2% FBS). Primary antibody was added at 1 μg/million cells in 100 μL total solution (for mouse cells: rat anti-mouse α_v integrin (BD Pharmingen 551380) or rat IgG isotype control (Invitrogen); for human cells: mouse anti-α_vβ₃ integrin, direct PE conjugate (BioLegend 304406) or mouse IgG κ chain isotype control, direct PE conjugate (BioLegend 400112); for neuropilin-1 staining in all cells, rabbit anti-NRP-1 (Novus Biologicals NBP1-40666) or normal rabbit IgG isotype control (R&D)) and incubated for one hour on ice. For direct fluorophore-conjugated primary antibodies, cells were washed with PBS and resuspended in FACS buffer. Otherwise, after washing the cells 2× in PBS, cells were incubated with secondary fluorescently-tagged antibody (Invitrogen) for 45 minutes and washed 1× in PBS. Cells were analyzed on BD LSR-II or Fortessa HTS flow cytometers. Data were analyzed in FlowJo (TreeStar Software).