Cd4 t cell biotin antibody cocktail

The spleens were isolated from the normal mice and homogenized as single cells in 6 ml Mouse 1×Lymphocyte Separation Medium (Dakewe biotech, China) in 15 ml tube. The cell suspension was centrifuged to separate the lymphocytes. The splenocytes in the middle layer were removed using a glass pipette, then washed with PBS and re-suspended in RPMI 1640 medium. The CD4⁺ T cells were isolated from the splenocytes using the CD4⁺ T cell isolation kit (Miltenyi Biotec, Germany) with CD4⁺ T cell biotin-antibody cocktail, anti-biotin microbeads and MACS columns (purity was > 95%).

CD4+ CD25− T cells were purified from healthy PBMCs by negative selection using a CD4+ T Cell Biotin-Antibody Cocktail (Miltenyi Biotec). Induced Treg (iTreg) cells were generated from CD4+ CD25− T cells of RA patients in the presence of an anti-CD3 antibody (eBioscience, San Diego, CA, USA), an anti-CD28 antibody (BD Pharmingen, San Diego, CA, USA), IL-2 (PEPROTECH), TGF-β (PEPROTECH), and vitamin D3 (SIGMA) [15 (link)]. CD4+ CD25− T cells from healthy controls were labeled for 10 min with 5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Eugene, OR, USA). RA iTreg cells and CFSE-labeled healthy effector T cells were co-cultured for 72 h at a ratio of 0:1, 0.3:1, and 1:1 in the presence of Dynabeads Human T-Activator CD3/CD28 (Invitrogen Dynal AS, Life Technologies, Oslo, Norway). T cell proliferation was measured by flow cytometry.

PBMC were washed in filter-sterilized PBS, 2 mM EDTA, 0.5% BSA (MACS buffer; 4 °C), and re-suspended in 90 μL of MACS buffer with 10 μL of CD4⁺ T Cell Biotin-Antibody Cocktail (Miltenyi Biotech) per 10⁷ total cells for 5 min at 4 °C. 20 μL of anti-biotin Microbeads (Miltenyi Biotech) per 10⁷ total cells was then added for 10 min at 4 °C, before increasing volume to 500 µl with MACS buffer. The cell suspension was added to a pre-rinsed LD column on a Midi-MACS separator magnet (Miltenyi Biotech). The column was washed with 1 ml of cold MACS buffer and the collected cells added to a second pre-rinsed LD column, this column was washed three times with 1 ml of cold MACS buffer to collect the unlabelled CD4⁺ fraction. CD4⁺ cells were washed and re-suspended in 90 μL of MACS buffer with 10 μL of CD25 Microbeads (Miltenyi Biotech) per 10⁷ total cells for 15 min at 4 °C, before washing in cold MACS buffer and being re-suspended in 500 μL of MACS buffer. The cell suspension was added to a pre-rinsed MS column on a Mini-MACS separator magnet (Miltenyi Biotech), and the column was washed three times with 500 µl of cold MACS buffer to collect the unlabelled CD4⁺ CD25⁻ cell fraction.