Macs ls separation column

10–14 week old C57BL/6 males served as gLN donors, and biological triplicates or quadruplicates were collected. Cells were sorted using a FACS Aria cell sorter flow cytometer (Becton Dickinson). MLN dendritic cells were pre-enriched using a Pan Dendritic Cell Isolation Kit (130–100-875, Miltenyi Biotec) and LS MACS Separation Columns (Miltenyi Biotec). Dendritic cells were sorted as Aqua^–CD45⁺Lin^–(CD3^–B220^–NK1.1^–CD19^–) CD11c^hi, and the subpopulations further as MHCII^hiCD103⁺CD11b^– and MHCII^hiCD103⁺CD11b⁺. Stromal cells were not pre-enriched and sorted as Aqua^–CD45^–TER119^–CD24^–TCRβ^–B220^–CD11c^– cells, and the subpopulations further as podoplanin⁺CD31^– (FRCs) and podoplanin⁺CD31⁺(LECs). Three hundred cells were sorted directly into 25 μl TCL buffer (Qiagen, 1031576) supplemented with 1% β-mercaptoethanol at single cell precision. Samples were kept at room temperature for 5 min, spun down and kept at −80 °C until further processing.

Genomic DNA was prepared from ear clips for PCR and sequence analysis. A 1–2 mm ear notch is placed in 50 μL alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA, pH ~12) and incubated at 95°C, overnight, before neutralizing with 50 μL of 40 mM Tris-HCl, pH ~5. Lymphocytes were prepared for analysis by multiple means. Spleens and bone marrow were harvested in IMDM supplemented with 5% FCS, 1 mM sodium pyruvate, 50 μg/mL gentamicin, 100 U/mL pen/strep, 2 mM l-glutamine, and 5 × 10^–5M beta-mercaptoethanol; single cell suspensions were prepared by mechanical disruption. RBCs were lysed with 1 mL of ACK (150 mM NH₄Cl, 10 mM KHCO₃, and 100 mM Na₂EDTA) for 1 min at room temperature (RT). Cells were subsequently washed and resuspended in complete medium.
For B cell isolation, spleen cells were harvested, RBC depleted by ammonium chloride tris lysis, and resuspended in 1 mL medium together with 40 μL of anti-CD43 Microbeads (Miltenyi Biotech) per mouse per spleen. The cell/bead mixture was rotated while incubating at 4°C for 10 minutes. LS MACS separation columns (Miltenyi Biotech) were washed with 5 mL medium. The cell/bead mixture was flowed over the column, which was then washed with 5 mL medium while collecting the flow-through. These preparations were routinely >95% B cells. Serum was prepared from peripheral blood collected from the tail vein.

Unmodified YT-1 (22 ) and IL-2Rα⁺ YT-1 human natural killer cells (23 (link)) were cultured in RPMI complete medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, minimum non-essential amino acids, sodium pyruvate, 25 mM HEPES, and penicillin-streptomycin [Gibco]) and maintained at 37ºC in a humidified atmosphere with 5% CO₂.
The subpopulation of YT-1 cells expressing IL-2Rα was purified via magnetic selection as described previously (24 (link)). Ten million unsorted IL-2Rα⁺ YT-1 cells were washed with FACS buffer (phosphate-buffered saline [PBS] pH 7.2 containing 0.1% bovine serum albumin) and incubated in FACS buffer with PE-conjugated anti-human IL-2Rα antibody (Biolegend, clone BC96) for 2 hr at 4°C. PE-labeled IL-2Rα⁺ cells were then incubated with paramagnetic microbeads coated with an anti-PE IgG for 20 min at 4º C, washed once with cold FACS buffer, and sorted on an LS MACS separation column (Miltenyi Biotec) according to the manufacturer’s protocol. Purified eluted cells were re-suspended and grown in RPMI complete medium. Enrichment of IL-2Rα⁺ cells was evaluated using an Accuri C6 flow cytometer (BD Biosciences) and persistence of IL-2Rα expression was monitored by PE-conjugated anti-human IL-2Rα antibody labeling and flow cytometric analysis of sorted IL-2Rα⁺ YT-1 cells.

Single cell suspensions were produced from pooled groups of two spleens, filtered, and incubated with anti-CD11c mAb-conjugated magnetic microbeads (Clone N418; Miltenyi Biotech) for 15 min in the dark at 4°C according to manufacturer’s instructions. Cells were washed and resuspended in cold Automacs buffer and passed through an LS MACS separation column (Miltenyi Biotech). Columns were washed three times with 3 mL of cold Automacs buffer and the positive fraction collected. Enriched cells were plated at 0.4-1 × 10⁶ cells/dish and stimulated for 7 h at 37°C with 1 μg/mL LPS (055:B5; Sigma-Aldrich) or 1 μg/mL CpG (ODN 1826; Integrated DNA Technologies). After 2 h of stimulation, 1 mg/mL of Brefeldin A (BD Biosciences) was added to each dish. After 7 h incubation, intracellular stains for IL-12/IL-23p40 (clone C15.6, Biolegend) and TNF-α (clone MPG-XT22, eBioscience) were performed after surface staining and fixation/permeabilization of the cell membrane using Cytofix/Cytoperm solution (BD Biosciences).