Human Ficoll–Hypaque density gradient centrifugation was employed to isolate peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample. To extracted CD4+T cells from PBMCs, CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were utilized using a procedure described previously in our laboratory.23 (link) Briefly, PBMCs (1 × 107) were incubated with 20 µL of CD4 microbeads and 80 µL of buffer solution at 4°C for 15 minutes. The cells were subjected to a mass spectrometry column (Miltenyi Biotec) filled with 500 µL of buffer solution. The cells were then collected following a buffer solution wash. CD4+T cells were cultured in exosome-free medium or Roswell Park Memorial Institute (RPMI) 1640 medium containing 100 U/mL penicillin/streptomycin and 10% fetal bovine serum and were then activated using 1 µg/mL anti-CD3 and 1 µg/mL anti-CD28 antibodies. Finally, they were treated with exosomes, miRNA mimic, miRNA inhibitor, or PBS. The samples were incubated for 24 hours or 72 hours at 37°C under 5% CO2 conditions, and cells or supernatants were finally collected for subsequent experiments.
Cd4 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, France
CD4 microbeads are a lab equipment product from Miltenyi Biotec. They are magnetic beads coated with antibodies specific to the CD4 receptor, a protein found on the surface of certain immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 microbead, by Miltenyi Biotec (18 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (8 mentions)
Ficoll paque plus, by GE Healthcare (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Ficoll paque, by GE Healthcare (6 mentions)
Cd19 microbeads, by Miltenyi Biotec (6 mentions)
Cd14 microbead, by Miltenyi Biotec (6 mentions)
Ls column, by Miltenyi Biotec (5 mentions)
Gentamicin, by Thermo Fisher Scientific (5 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (5 mentions)
Cd3 microbeads, by Miltenyi Biotec (5 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (5 mentions)
Facsaria 3, by BD (4 mentions)
Lsr 2, by BD (4 mentions)
Trypan blue, by Thermo Fisher Scientific (4 mentions)
Ms column, by Miltenyi Biotec (4 mentions)
Facscalibur, by BD (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Cd11c microbeads, by Miltenyi Biotec (3 mentions)
226 protocols using cd4 microbeads
1
Isolation and Activation of CD4+ T Cells
2
Adoptive Transfer of CD4+ T Cells
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Purified CD4+ T cells (5 × 106) with CD4 microbeads ((Miltenyi Biotec) were transferred into unirradiated C57BL/6 mice. In some experiments, mice were immunized with OVA protein (50 μg/mouse) emulsified in complete Freund’s adjuvant (SLBD0737, Sigma). In some experiments, T cells were stimulated with anti-CD3 mAb (1 μg/ml) and infected two times with retrovirus, as previously reported10 (link). The infection efficiency was similar (30~40%) between control virus and Dll1-carrying virus (data not shown). GFP-positive cells were purified four days after stimulation by cell sorting (FACSAria III, BD Biosciences) for transfer into recipient mice. For in vitro T cell survival assay, purified CD4+ T cells by CD4 microbeads (Miltenyi Biotec) from Dll1−/−: OT-II (Thy1.1−Thy1.2+) or Dll1+/+: OT-II (Thy1.1+ Thy1.2+) mice were stimulated with irradiated spleen cells from C57BL/6 mice (Thy1.1+ Thy1.2−) and OVA protein (50 μg/ml). The ratio of Thy1.1+ and Thy1.1− cells gated on Thy1.2+ cells were evaluated.
3
Th1, Th2, and Th17 Polarization of Lymph Node Cells
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Lymph node cells were cultured for 3 days in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 5 mM l-glutamine, 50 μM 2-ME, and antibiotics (Penicillin-Streptomycin) in the presence of 20 μg/ml MOG35–55 peptide under Th1 (IL-12 10 μg/ml and anti-IL-4 10 μg/ml), Th2 (IL-4; 30 ng/ml and anti-IFN-γ; 10 μg/ml) or Th17-polarizing (IL-6; 10 ng/ml, TGF-β1; 1 ng/ml, anti-IFN-γ; 10 μg/ml and anti-IL-4; 10 μg/ml) conditions. T cells purified by CD4 microbeads and LS columns (Miltenyi Biotec) were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 hours to perform ELISA assay. GeneJuice (Merck Millipore) was used to generate retroviruses by transfecting each vector encoding GFP into Plat-E cells (provided by Dr. T. Kitamura)25 (link). T cells purified by CD4 microbeads and LS columns (Miltenyi Biotec) were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 hours. After the addition of supernatant containing retroviruses carrying Dll1 or the intracellular domain of Dll1, cells were centrifuged at 2600 rpm for 1 hour two times at 8-hour intervals.
4
Isolation and Purification of CD4+ T Cells
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The collected spleen was placed into a 5 ml EP tube lled with cold phosphate buffer saline (PBS). Then, the spleen was minced and ground with a syringe piston in a 70 µm sieve to produce a single cell suspension. After centrifugation at 1500 rpm for 5 minutes, the isolated splenocytes were resuspended with PBS. The splenocytes suspension was carefully added to the upper layer of an equal amount of lymphocyte separation solution with a pipette, and the mixed solution was centrifuged at 3000 rpm for 20 minutes. Then, the cloudiness ring-shaped lymphocyte in the middle layer was carefully pipetted into a new 15 ml centrifuge tube, and wash three times with PBS. After cell counting, resuspended each 10 7 cells with 80 µl PBS were mixed with 20 µl of CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for incubation for 15 minutes at 4 °C. The CD4 MicroBeads labeled CD4 + T cells were obtained by a separation column (Miltenyi Biotec) in the magnetic eld of a MACS separator. After washing and centrifugation, the nal cell concentration was determined to be 2 × 10 6 cells / ml and then resuspended in RPMI 1640 containing 10% heat-inactivated fetal bovine serum and 1% antibiotic at 37 °C.
5
Comprehensive Immune Cell Profiling
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Peripheral blood mononuclear cells isolated by Ficoll gradient separation were split into CD4+ and CD4− fractions using magnetic bead separation (Miltenyi, CD4 Microbeads). Genomic DNA was isolated from both fractions and used to generate quantitative TCRβ, TCRδ, and immunoglobulin-heavy chain libraries from a single-well assay using the immunoPETE protocol from Roche Sequencing Solutions. The immunoPETE protocol combines V gene priming and extension with UMI-tagged oligos, followed by J-primer extension and library amplification of bead-purified PCR products from the initial V primed extension. The full experimental protocol is provided in the SI Appendix, Materials and Methods .
6
Single-cell flow cytometry protocol
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Single cell suspension was first stained with the fixable viability dye at 1:1000 in PBS for 10 min. After washing with flow activated cell sorting (FACS) buffer (PBS/2%FBS), cells were incubated with Fc block at 1:200 for 10 min, followed by staining with indicated antibody mixtures for 30 min before washing and flow cytometry analysis. For intracellular staining, including IgE, cells were fixed and permeabilized using the FoxP3 staining Buffer Set according to the manufacturer’s protocol. Cells were then incubated with Fc block and intracellular antibodies for 30 min before washing and flow cytometry analysis. All of the steps were performed at 4 °C. For intracellular cytokine analysis, cells were stimulated with the BD Leukocyte Activating Cocktail, with BD GolgiPlug for 5 h at 37 °C with 5% CO2, prior to staining, as described above. Cells were acquired on a BD LSR II or FACSymphony using FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Treestar). For cell sorting, single cell suspensions isolated from spleens were enriched for CD4+ T-cells using the CD4 microbeads (Miltenyi Biotec). Enriched CD4+ T-cells or tumor cells were then stained with viability dye and surface antibodies as described above, followed by sorting on a FACSAria II using FACSDiva software.
7
CD4+ T Cell Activation and Isolation
8
Isolation and Purification of CD4+ T Cells
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Blood mononuclear cells were isolated from the samples of the femoral artery and peripheral blood of LEASO patients and HCs. Whole blood containing an acid citrate dextrose anticoagulant (Biological Specialty Corporation, Colmar, PA, USA) was carefully layered over an equal volume of Ficoll (Shanghai, China; ρ=1.077 ± 0.002 g/ml) density gradient medium. Tubes containing the blood and Ficoll were centrifuged for 20 min at 2,000 rpm. Isolated arterial blood mononuclear cells (ABMCs) and peripheral blood mononuclear cells (PBMCs) were washed two times in phosphate-buffered saline (PBS) and centrifuged for 10 min at 1,200 rpm and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B at 37°C in 95% air and 5% CO2. CD4+ T cells were isolated by magnetic affinity cell sorting using CD4 microbeads (Miltenyi Biotec, Auburn, CA, USA) with LS columns following the manufacturer’s instructions.
9
PBMC Isolation by Density Gradient
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Peripheral blood mononuclear cells (PBMCs) were isolated by standard density gradient centrifugation with either Lymphocyte Separation Buffer (Anprotec) or Ficoll Paque (GE). Where indicated, CD4+ T cells were purified from PBMCs with CD4 microbeads (Miltenyi).
10
Differentiation and Activation of T Cell Subsets
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A single cell suspension was isolated from spleen and lymph nodes of the mice as previously described (Lee et al., 2013 (link)). Lymph node cells of the OT-II mice were activated with 0.2 μg/ml OVA323–339 peptide in Iscove’s Modified Dulbecco’s medium containing 10% FBS and cultured with 4 ng/ml IL-2 for 5 days. CD4+ T cells were purified from the spleen of C57BL/6J mice using CD4 microbeads (Miltenyi Biotech, Gladbach, Germany) in accordance with the manufacturer’s instructions. CD4 T cells were activated with anti-CD3ε (2.5 μg/ml) and anti-CD28 (2.5 μg/ml) and cultured for 5 days with mitomycin C-treated splenocytes. Th17 culture was supplemented with 5 ng/ml TGF-β, 10 ng/ml IL-6, 10 ng/ml IL-23, 5 μg/ml anti-IL-12, 5 μg/ml anti-IFN-γ and 5 μg/ml anti-IL-4.
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