Naïve CD4+ T cells were obtained by negative selection using the CD4+ T‐Cell Isolation Kit II (Miltenyi Biotec) from stimulated PBMCs. Nontarget cells were labeled with a cocktail of biotin‐conjugated monoclonal antibodies (MicroBead Cocktail, Miltenyi Biotec), and the magnetically labeled nontarget T cells were retained on a column in the magnetic field of a separator (Miltenyi Biotec). This protocol produces > 95% pure CD4+ T cells, as tested by fluorescence‐activated cell sorting analysis. Cells were resuspended at 2 × 106 cells/mL in RPMI‐1640 culture medium (Gibco) supplemented with 10% FBS, penicillin/streptomycin (1%) (Lonza), L‐glutamine (1%), sodium pyruvate (1%) (Lonza), and NEAA (1%) (Lonza). Cells were cultured at 37°C in complete medium at concentrations of 2x105 cells/ml in 24‐well plate (Nunc). Naïve CD4+ T cells obtained were processed for flow cytometry assays and for DNA/RNA extraction. All experiments were performed in triplicate and repeated twice.
Microbead cocktail
Manufactured by Miltenyi Biotec
The MicroBead Cocktail is a laboratory equipment product from Miltenyi Biotec. It is a suspension of magnetic beads that can be used for the isolation or detection of specific cell types or molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Sodium pyruvate, by Lonza (1 mentions)
Cd11c microbeads ultrapure, by Miltenyi Biotec (1 mentions)
Anti cd16 32 antibody, by Thermo Fisher Scientific (1 mentions)
2 protocols using microbead cocktail
1
Isolation and Culture of Naïve CD4+ T Cells
2
Isolation and Stimulation of CD11b+ Myeloid DCs
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To manipulate CD11b+ myeloid DCs ex vivo, spleens were harvested from naive or LCMV-infected (10 days postinfection) mice. The spleens were enzymatically digested with 1 mg/ml type II collagenase D and 1 mg/ml DNase I. Red blood cells were lysed with ACK lysis buffer. CD19+, CD49b+, and CD90.2+ populations were primarily removed with a microbead cocktail (Miltenyi Biotec), and CD16/32 was blocked with a purified anti-CD16/32 antibody (Thermo Fisher). Then, CD11c+ cells were isolated with CD11c MicroBeads UltraPure (Miltenyi). For stimulation of DCs, 500 ng/ml LPS was added to the culture medium for 5 hours, and the stimulated DCs were cocultured with CellTrace™ Violet (Thermo Fisher)-labeled CD8+ P14 cells, OT-I T cells or CD4+ T cells.
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