Naïve CD4+ T cells were isolated from fresh human blood samples with a negative selection isolation kit from Miltenyi Biotec as per protocol. The purity of naïve CD4+ T cells was evaluated with staining of anti-CD3 (Clone UCHT1, BioLegend, San Diego, USA), anti-CD4 (clone RPA-T4, BioLegend, San Diego, USA), and anti-CD45RA (HI100, BioLegend, San Diego, USA). Isolated cell purities were over 95% (Supplementary Figure S1 ). Cells were cultured in RPMI 1640 media (GE Health Care Life Sciences, Marlborough, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, USA). Cells were stimulated overnight with anti-CD3 (10 μg/mL, pre-coated on plate, Clone UCHT1, BD Biosciences, San Jose, USA) and anti-CD28 (2.5 μg/mL, Clone CD28.2, BD Biosciences, San Jose, USA), with and without glycolysis inhibitor 2-deoxy-d -glucose (2-DG, 2 mg/mL, Acros Organics, New Jersey, USA), mTOR inhibitor rapamycin (100 Nm, Alfa Aesar, Ward Hill, USA) or H2O2 (50 μM, Sigma-Aldrich, St. Louis, USA). The next day the antibodies were removed and fresh media (RPMI and 10% FBS) were added with and without 2-DG, rapamycin, or H2O2. The cells were cultured for a total of 3 days before harvesting.
Negative selection isolation kit
Manufactured by Miltenyi Biotec
The Negative selection isolation kit is a lab equipment product designed to isolate specific cell types from a mixed cell population. The kit utilizes an antibody-based approach to negatively select the desired cells, leaving the target cells unaltered for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 media, by GE Healthcare (2 mentions)
Anti cd3 clone ucht1, by BioLegend (2 mentions)
Clone cd28, by BD (2 mentions)
Anti cd4 clone rpa t4, by BioLegend (2 mentions)
Clone ucht1, by BD (2 mentions)
Mtor inhibitor rapamycin, by Thermo Fisher Scientific (2 mentions)
2 deoxy d glucose 2 dg, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using negative selection isolation kit
1
Activation and Metabolic Regulation of Naive T Cells
2
Purification and Transfer of Naive CD8 T Cells
3
Activation and Metabolic Regulation of Naive T Cells
4
CD4 T-cell Isolation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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About 10 ml fresh blood was collected from each MS patient or healthy donor and centrifuged. After lysis of the red blood cells, total white blood cells were subjected to CD4 T-cell isolation using a negative selection isolation kit (Miltenyi Biotech, San Diego, CA) following the instruction. Purified CD4 T cells were utilized for the following experiments: for RNA purification and real-time PCR analysis, isolated cells were suspended in Trizol reagent and total RNA was isolated according to the manufacturer's instructions (Invitrogen). For the shRNA knockdown experiment, CD4 T cells were infected with lentivirurs carrying Hrd1-specific or control shRNA. Green fluorescent protein-positive cells were either gated or sorted for analysis by intracellular staining or western blotting. The study has been approved by the Institutional Review Board.
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