Pre-immune serum was obtained from each mouse a week before immunization. ATX mice were successively immunized with recombinant PDCoVIL121_2014 RBD and S diluted (1:1) in Magic Mouse adjuvant (Cat#: CDN-A001E; CD Creative Diagnostics) and injected subcutaneously. On day 0, mice received prime immunization with 5 μg of PDCoV RBDIL121_2014 and were boosted on day 14 with 5 μg of PDCoV SIL121_2014 ectodomain. On day 29, the mice received 5 μg of PDCoV RBDIL121_2014 and on day 56 they received a last boost with 2 μg of PDCoV SIL121_2014. On day 64, the mice were sacrificed and peripheral blood, spleen and lymph nodes (LN) were collected and cells freshly isolated. B cells were enriched by positive selection using mouse CD19 microbeads and LS columns (Miltenyi). Enriched B cells were then stained with mouse anti-IgM, anti-IgD, and biotinylated PDCoV SIL121_2014 ectodomain labeled with both PE and Alexa-Fluor 647 streptavidin (Life Technologies). Sorted IgG+ memory B cells were seeded at clonal dilution in 384-well plates an a monolayer of feeder mesenchymal cells in the presence of B cell survival factors. Clones positive for antigen binding were then isolated, sequenced and produced recombinantly in transiently transfected CHO cells.
Mouse cd19 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Mouse CD19 MicroBeads are magnetic beads coated with antibodies specific for the CD19 surface antigen on mouse B cells. They can be used for the isolation or depletion of CD19+ B cells from cell suspensions.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Easysep mouse cd4 t cell isolation kit, by STEMCELL (1 mentions)
As01b, by GlaxoSmithKline (1 mentions)
Ovalbumin (ova), by Worthington (1 mentions)
Anti mouse igg hrp, by Fortis Life Sciences (1 mentions)
Mouse igg elisa quantitation set, by Fortis Life Sciences (1 mentions)
El800, by Agilent Technologies (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
30 m cell strainer, by Miltenyi Biotec (1 mentions)
Mouse cd4 t cell isolation kit, by STEMCELL (1 mentions)
11 protocols using mouse cd19 microbeads
1
Isolation and Characterization of PDCOV-Specific Memory B Cells
2
Mouse Dendritic Cell and T/B Cell Isolation
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Mouse bone marrow-derived dendritic cells (mBMDCs) were generated from the femurs and tibias of mice as previously described (15 (link)). Mouse CD4+ T cells and CD19+ B cells were isolated from splenocytes using EasySep Mouse CD4 T Cell Isolation Kit (ST-19852, STEMCELL Technologies, Vancouver, Canada) and mouse CD19 MicroBeads (130-121-301, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Cells were cultured in RPMI supplemented with 10% FBS (Omega, Tarzana, CA, USA) and penicillin/streptomycin (100 unit/mL/100 μg/mL, Thermo Fisher Scientific, Waltham, MA). Compound 2D216 was synthesized in our laboratory, and purity was confirmed by LC/MS. Ovalbumin (OVA) was purchased from Worthington Biochemical (Lakewood, NJ, USA). LPS (LPS-EB Ultrapure tlrl-eblps) and MPLA (vac-mpla) were purchased from InvivoGen (San Diego, CA, United States). AS01B was purchased from GlaxoSmithKline (GSK, Middlesex, United Kingdom, Zoster Vaccine Recombinant, Adjuvanted SHINGRIX).
3
Isolation and Expansion of CD19+ TILs for IgG Detection
4
Isolation of Primary Mouse B Cells
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Primary mouse B cells were isolated from the spleen of 8-week-old to 12-week-old C57BL/6 mice (males or females, bred in-house). Spleens were mechanically dissociated and passed through a 70 μm cell strainer (Corning, Sigma-Aldrich, St. Louis, MO, USA) to obtain a single-cell suspension in sterile PBS. Splenic B cells were isolated by positive selection using Mouse CD19 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions, and were confirmed ≥98% positive for B220 by flow cytometry.
5
Isolation and Stimulation of CD19+ B Cells
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For in vitro experiments, CD19+ B cells were isolated from spleens using mouse CD19 microbeads (Miltenyi Biotec). Then, B cells were cultured in medium supplemented with 10 µg/ml LPS (Sigma-Aldrich) alone or together with G-exo.
6
Isolation of Murine T and B Cells
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Splenocytes were counted under a microscope, and diluted to a final concentration of 2×106 cells/ml for cell culture. CD19+ B cells were sorted using mouse CD19 Micro-beads (Miltenyi Biotech, 130-052-201) according to the manufacturer’s instructions. CD3+ T cells were stained by FITC labeled anti-mouse CD3 antibody, and sorted by FCM (moflo, Beckman coulter, USA). The purity of T and B cells was above 90%, which were used immediately after sorting.
7
Gut Microbiota-Priming of iNKT Cells
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Detection of IL8/CXCL8 in culture supernatants was performed using the OptEIA Human IL-8 kit (BD Biosciences, IT), according to the manufacturer's instruction.
Gut microbiota-priming of murine iNKT cells Splenic iNKT cells were isolated from C57BL/6 mice sorting CD45 + CD3 + CD1d:PBS57Tet + cells upon enrichment through B cells exclusion (Mouse CD19 microbeads, Miltenyi Biotec, Germany). Bone marrow-derived dendritic cells from C57BL/6 mice were pulsed with heat-inactivated fecal microbiota of controls or AOM-DSS treated C57BL/6, Traj18 -/-, and CD1d -/-mice and cocultured with freshly isolated splenic iNKT cells (2 × 10 5 cells) in a 2:1:10 iNKT:bone marrow-derived dendritic cells:microbiota ratio in RPMI-1640 supplemented with 10% FBS and Pen/Strep solution. After 24 hours, the iNKT cell activation status was estimated by intracellular staining. Fecal samples were resuspended 1:10 (w/v) in PBS and filtered through a 0.75-μm filter to remove large debris; microbiota cell density was quantified by quantitative Polymerase Chain Reaction (qPCR) 48 , adjusted to 2 × 10 7 CFU•mL-1, and heat-killed at 95°C for 15 minutes before being stored at -80°C until use in downstream experimentation.
Gut microbiota-priming of murine iNKT cells Splenic iNKT cells were isolated from C57BL/6 mice sorting CD45 + CD3 + CD1d:PBS57Tet + cells upon enrichment through B cells exclusion (Mouse CD19 microbeads, Miltenyi Biotec, Germany). Bone marrow-derived dendritic cells from C57BL/6 mice were pulsed with heat-inactivated fecal microbiota of controls or AOM-DSS treated C57BL/6, Traj18 -/-, and CD1d -/-mice and cocultured with freshly isolated splenic iNKT cells (2 × 10 5 cells) in a 2:1:10 iNKT:bone marrow-derived dendritic cells:microbiota ratio in RPMI-1640 supplemented with 10% FBS and Pen/Strep solution. After 24 hours, the iNKT cell activation status was estimated by intracellular staining. Fecal samples were resuspended 1:10 (w/v) in PBS and filtered through a 0.75-μm filter to remove large debris; microbiota cell density was quantified by quantitative Polymerase Chain Reaction (qPCR) 48 , adjusted to 2 × 10 7 CFU•mL-1, and heat-killed at 95°C for 15 minutes before being stored at -80°C until use in downstream experimentation.
8
Isolation of Murine B and T Cells
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Spleens from mice of WT and BKO groups were cut into pieces and gently grounded with a grinding rod. Single cell suspensions were ltered through a 30 µm cell strainer (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells from WT group VMC mice were isolated using mouse CD19 MicroBeads (Miltenyi Biotec), following the manufacturer's instructions (catalog #130-052-021). Single cell suspensions from mice of WT and BKO groups were prepared as described above to isolate naïve CD4 + T cells. Naive CD4 + T cells were isolated with the speci c naïve CD4 + T cell isolation kit (Miltenyi Biotec) following to the manufacturer's instructions (catalog #130-104-453). The purity of B cells or naïve CD4 + T cells was determined by ow cytometry to be >97%.
9
Soluble CD83 Production in Lymphocytes
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To determine which cells produce soluble CD83, splenic lymphocytes were depleted from CD4+ or CD19+ cells. CD19 depletion was performed using CD19 MicroBeads mouse (Miltenyi Biotec GmbH, Teterow, Germany). For CD4 depletion, a negative selection Mouse CD4+ T-Cell Isolation kit was purchased from EasySep (STEMCELL Technologies, Vancouver, BC, Canada). In both cases, the supplier’s instructions were followed; 500,000 cells were cultured with 250 µL culture medium and stimulated with LPS as described before.
10
Splenic Leukocyte Isolation and Stimulation
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Whole splenic leukocytes were isolated using standard laboratory procedures described in detail previously9 (link),38 (link),40 (link),49 (link),60 (link),61 (link). Briefly, the spleens were dissociated by gently scraping through a steel screen, and the cell suspension was passed through a 70-μm cell strainer to remove tissue debris. To eliminate the unintended in vitro exposure to estrogens, care was taken to use only charcoal-stripped FBS and phenol red-deficient media. The splenic leukocytes were adjusted to 5×106/ml in complete medium for seeding into cell culture plates for stimulation. Briefly, the cells were plated into 48-well cell culture plates (0.25 ml/well), and stimulated with Imiquimod (5 µg/ml) (IDT Inc., Skokie, IL, USA), ODN-2395 (0.5μM, synthesized by IDT Inc.), ODN Control (IDT Inc.), or complete media for the designated time by adding an equal volume of 2x concentration of stimulation medium. CD4+ and CD19+ cell were purified from whole splenic leukocytes using the Miltenyi Biotec “CD4+ (L3T4) MicroBeads, mouse,” and “CD19+ MicroBeads, mouse,” per the company’s published manual separation protocol (Miltenyi Biotec Inc., Auburn, CA, USA). Cell pellets were washed with cold PBS and stored at −80 °C.
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