Hapten-specific B cells were enriched from the spleens of B1–8+ Jκ-/- mice by immunomagnetic purification with the EasySep Negative Selection Mouse B Cell Enrichment Kit (StemCell Technologies). T cells were isolated from the spleens and lymph nodes of OT-II mice with the EasySep Negative Selection Mouse CD4+ T Cell Enrichment Kit (StemCell Technologies). For all flow cytometry, sorting and histology experiments, 3 × 106 B cells and 1 × 106 T cells were transferred prior to immunization. To ensure consistency in the cell transfer of the donor cell populations, all recipients of a time course experiment received their transferred cells on the same day. Cells were injected intravenously into SMARTA recipients that were immunized 1 – 5 days later such that the tissue harvest, staining and flow cytometric analysis for the entire time course study were begun on the same day. In some experiments, wild type C57BL/6 mice were used as recipients instead. For immunizations, NP-OVA was precipitated in alum at 0.25 mg/mL and 50 μg injected i.p. The succinic anhydride ester of Nitrophenyl (Biosearch Technologies) was conjugated to ovalbumin (Sigma) in-house. In some transfer studies, 3mg BrdU in 200 μL PBS was injected I.V. in the tail vein 6 hrs before sacrifice.
Easysep negative selection mouse cd4 t cell enrichment kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep Negative Selection Mouse CD4+ T Cell Enrichment Kit is a laboratory equipment product that enables the isolation of mouse CD4+ T cells from a single-cell suspension. The kit employs a magnetic bead-based separation technique to negatively select the target cells, leaving them untouched for downstream applications.
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Lab products found in correlation
Ovalbumin (ova), by Merck Group (1 mentions)
Easysep negative selection mouse b cell enrichment kit, by STEMCELL (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Cellection pan mouse igg kit, by Thermo Fisher Scientific (1 mentions)
Josamycin, by Astellas Pharma (1 mentions)
2 protocols using easysep negative selection mouse cd4 t cell enrichment kit
1
Adoptive Transfer to Analyze B-T Cell Interactions
2
Mast Cell-Th Cell Interaction Assay
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Mast cells were adjusted to 1×10 5 cells/mL in RPMI 10 and incubated in the presence or absence of PEG (10 g/mL) and josamycin (10 M;
provided by Astellas Pharma Inc., Tokyo, Japan) for 18h at 37C in a humidified atmosphere with 5% CO2, and then washed before use. Next, T helper (Th) cells were separated from DO 11.10 TCR Tg mouse spleen cells using an EasySep Negative Selection Mouse CD4 + T Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and then treated with mouse anti-CD62L monoclonal antibody (clone lam1-116, IgG2a) (1 g per 1 × 10 6 cells; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in RPMI 10 for 1 h on ice. The Th cells that had been reacted with anti-CD62L antibody were then purified using a CELLection TM Pan Mouse IgG Kit (Invitrogen Dynal AS, Oslo, Norway), and used as naïve Th cells. The naïve Th cells (5 × 10 5 cells/mL) were cultured with the above mast cells (1 × 10 5 cells/mL) in the presence of 30 nM OVA peptide (323-ISQAVHAAHAEINEAGR-339; obtained from Operon Biotechnologies, Tokyo, Japan) for 5 days at 37°C. The cells were then stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) and 500 ng/mL ionomycin (Sigma) for 24 h at 37°C. The cell supernatants were finally removed and tested for production of interferon (IFN)- and IL-4 using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).
provided by Astellas Pharma Inc., Tokyo, Japan) for 18h at 37C in a humidified atmosphere with 5% CO2, and then washed before use. Next, T helper (Th) cells were separated from DO 11.10 TCR Tg mouse spleen cells using an EasySep Negative Selection Mouse CD4 + T Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and then treated with mouse anti-CD62L monoclonal antibody (clone lam1-116, IgG2a) (1 g per 1 × 10 6 cells; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in RPMI 10 for 1 h on ice. The Th cells that had been reacted with anti-CD62L antibody were then purified using a CELLection TM Pan Mouse IgG Kit (Invitrogen Dynal AS, Oslo, Norway), and used as naïve Th cells. The naïve Th cells (5 × 10 5 cells/mL) were cultured with the above mast cells (1 × 10 5 cells/mL) in the presence of 30 nM OVA peptide (323-ISQAVHAAHAEINEAGR-339; obtained from Operon Biotechnologies, Tokyo, Japan) for 5 days at 37°C. The cells were then stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma) and 500 ng/mL ionomycin (Sigma) for 24 h at 37°C. The cell supernatants were finally removed and tested for production of interferon (IFN)- and IL-4 using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).
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