Magnetized ls column

For phenotyping B cells, 6-week old female BALB/c mice, three per dosing group, were immunized intramuscularly with 50 μL per injection site of immunogen formulations mixed 1:1 (vol/vol) with AddaVax adjuvant on day 0. All experimental mice were euthanized for harvesting of inguinal and popliteal lymph nodes on day 11. The experiment was repeated twice. Popliteal and inguinal lymph nodes were collected and pooled for individual mice. Cell suspensions were prepared by mashing lymph nodes and filtering through 100 μm Nitex mesh. Cells were resuspended in PBS containing 2% FBS and Fc block (2.4G2), and were incubated with 10 nM decoy tetramers at room temperature for 20 min. I53_dn5A-PE tetramer and HA-APC tetramer, or I53_dn5B-PE tetramer and HA-APC tetramer, were added at a concentration of 10 nM and incubated on ice for 20 min. Cells were washed, incubated with anti-PE and anti-APC magnetic beads on ice for 30 min, then passed over magnetized LS columns (Miltenyi Biotec). Bound B cells were stained with anti-mouse B220 (BUV737), CD3 (PerCP-Cy5.5), CD138 (BV650), CD38 (Alexa Fluor 700), GL7 (eFluor 450), IgM (BV786), IgD (BUV395), CD73 (PE-Cy7), and CD80 (BV605) on ice for 20 min. Cells were run on a Cytek Aurora and analyzed using FlowJo software (Treestar). Cell counts were determined using Accucheck cell counting beads.

Brains harvested for microglial extraction were placed in cold HBSS and diced before processing immediately. Brains were dissociated using a GentleMACS dissociator (Miltenyi Biotec) and a neural tissue dissociation kit P (Miltenyi Biotec). The final cell pellet was resuspended in 16 ml of 35% Isotonic Percoll, split between two 15-ml tubes, and carefully overlaid with 5 ml of ice-cold 0.1% diethyl pyrocarbonate (DEPC)-treated HBSS. The resulting Percoll gradient was centrifuged at 400 × g for 45 min at 4°C. The pellets were then suspended and recombined into a final volume of 5 ml ice cold 0.1% DEPC-treated HBSS. Cells were pelleted at 400 × g for 5 min at 4°C using no brake, resuspended in 90 μl of ice-cold MACS buffer (Miltenyi Biotec) and 10 μl of CD11b (microglia) microbeads (Miltenyi Biotec), and incubated at 4°C for 15 min with gentle rotation. After incubation with microbeads, the cell suspension was washed in 1 ml of ice-cold MACS buffer at 300 × g for 5 min at 4°C and then resuspended in 500 μl of ice-cold MACS buffer. Cells were passed through magnetized LS columns (Miltenyi Biotec) according to the manufacturer's protocol.

Purified CD4+ T cells were obtained from spleen or dLN using magnetic bead separation (Miltenyi Biotec). 2W:I-Ab tetramer staining and magnetic enrichment were performed as previously described [31] (link). Briefly, a single cell suspension of the spleen, dLN, and ndLNs from a single mouse were stained with 10 nM allophycocyanin- or phycoerythrin-labeled 2W:I-Ab-streptavidin tetramers for 1 hour at room temperature. Samples were then chilled to 4°C and incubated with magnetic anti-fluorochrome beads and run through a magnetized LS column (Miltenyi Biotec) in a 4°C cold room. To determine the absolute number of cells, a portion of each sample was removed for counting with AccuCheck Counting Beads (Invitrogen) as described previously [31] (link).

Samples were incubated with 5nM Decoy Alexa Fluor 647 conjugated to PE (AF647-PE) at room temperature for 5 min as described [20] . Then samples were incubated with 5 nM 6OXY-PE for 25 min at +4°C. Samples were washed in ice-cold sorter buffer and resuspended to a final volume of 200 µl using sorter buffer, then mixed with 25 µl of anti-PE conjugated magnetic beads (Miltenyi Biotech, Inc, Auburn, CA) and incubated for 15 min at +4°C. The cells were resuspended in sorter buffer and passed through a magnetized LS column (Miltenyi Biotech). For each sample, bound fractions containing B cells bound to PE and 6OXY-PE were centrifuged at 1600 RPM for 5 min at +4°C and resuspended in a final volume of 100 µl of sorter buffer. From each sample, 5 µl were added to 200 µl of lymphocyte fluorescent counting beads at a concentration of 200,000 beads/ml (Accucheck, Invitrogen, Frederick, MD) to calculate numbers of hapten-specific B cells in each sample as described [20] .

To determine the endogenous naive precursor frequency of MCMV-specific CD8⁺ T cell populations in C57BL/6 and BALB/c mice, enrichment assays of antigen-specific CD8⁺ T cells were performed as described [42 (link)]. In short, single cell suspensions were generated from pooled spleen and lymph nodes (mesenteric, inguinal, cervical, axillary, and brachial) of individual mice. Cells were stained with PE and APC-labelled MHC class I tetramers for 0.5 h at RT, then washed, labelled with anti-PE and anti-APC microbeads (Miltenyi Biotec), and passed over a magnetized LS column (Miltenyi Biotec). The tetramer-enriched fractions were stained with fluorochrome labelled Abs against CD3 (clone 500A2), CD4 (clone L3T4), CD8 (clone 53–6.7) for 30 min at 4°C, and subsequently analysed. Samples were acquired with the LSRFortessa cytometer (BD Biosciences).