Mouse BM cells were flushed from femurs and tibias with Dulbecco medium. Erythrocytes were lysed with lysis buffer (eBioscience) for 3 min at room temperature, and the remaining cells were washed once with PBS. Single cell suspensions were isolated from spleens and erythrocytes were lysed with lysis buffer. MDSCs were isolated from splenocytes by magnetic cell separation (Miltenyi, Germany). Flow cytometric analysis revealed high purity (90%) of isolated CD11b+Gr-1+ cells. CD4+ cells were isolated by magnetic cell separation using the CD4+ T cell isolation kit (Miltenyi), while CD4+CD25+ Treg cell isolation kits (Miltenyi) were used to isolate CD4+CD25− cells and perform adoptive transfer colitis.
Magnetic cell separation
Manufactured by Miltenyi Biotec
Sourced in Germany
Magnetic cell separation is a lab equipment product that enables the separation and isolation of specific cell types from a heterogeneous cell population. It utilizes paramagnetic beads coated with antibodies or ligands that bind to target cell surface markers, allowing for the selective capture and separation of the desired cell population using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 3 (il 3), by Thermo Fisher Scientific (3 mentions)
Cpd celltrace violet, by Thermo Fisher Scientific (2 mentions)
Iniscove s modified eaglemedium, by Corning (2 mentions)
Flt3l, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Cd4 cd25 treg cell isolation kit, by Miltenyi Biotec (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Hydrocortisone, by Thermo Fisher Scientific (1 mentions)
G csf, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide, by InvivoGen (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti biotin magnetic beads, by Miltenyi Biotec (1 mentions)
Polyinosinic polycytidylic acid poly 1 c, by InvivoGen (1 mentions)
Imject alum, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Fcs gold, by Bio&Sell (1 mentions)
Cetuximab, by Bristol-Myers Squibb (1 mentions)
26 protocols using magnetic cell separation
1
Isolation of Mouse Myeloid-Derived Suppressor Cells
2
Evaluation of CMV-specific CD4+ T cells
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CD4+ T cells were isolated from HLA-A*0201+ PBMCs using magnetic cell separation (Miltenyi Biotech) and cultured for 6 hours (1 x 106 cells/ml in RPMI10 containing 20μg/ml DNase), to allow Tax expression. The cells were then loaded with the CMV peptide pp65 (Think Peptides) at a range of concentrations (0–2 μM). CMV-specific CTLs were added to the peptide-loaded CD4+ cells at an effector:target ratio of 1:1 and co-cultured for 12 hours. All samples were assayed in duplicate. A small portion of cells was used to determine absolute cell counts and the remainder were stained with a viability stain, anti-CD3-BV510, -CD4-BV605, -CRTAM-PE, -CD8-AF700, -CADM1–Biotin and -Tax-Cy5.
3
Long-term Expansion of CD34+ Blasts
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Following positive isolation using magnetic cell separation (Miltenyl Biotec Inc, Germany), 5×104 CD34+ blasts were cultured on MS5 stromal cells and expanded in hematopoietic media (Myelocult/Stemcell, Grenoble, France) containing 1 mM hydrocortisone, IL-3, GCSF and TPO (20 ng/ml) (Peprotech, Rocky Hill, NJ, USA). Following weekly demi-depopulation, long-term cultures were established from 8 of 11 patient samples as previously described [15] (link), [16] .
4
Th1/Th2 Immune Response and N. brasiliensis Infection
5
Expanding and Transducing Pro B-cells
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B220+ cells were isolated from bone marrow of ER-Cre mice (55 (link)) using magnetic cell separation (Miltenyi Biotec), expanded for 6 days in the presence of IL-7 and SCF to obtain pro B-cells. Pro B-cells were subsequently retrovirally transduced with Bcl-2, expanded for seven additional days with 5 μM 4-Hydroxytamoxifen in the medium during the last 72 h. Pro B-cells were fixed using EGS (1.5 mM for 30 min) in combination with PFA (1% for 10 min) and stored as pellets in −80°C.
6
Activation and Differentiation of Mouse CD4+ T Cells
7
Isolation of Equine Immune Cells
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Different equine tissue samples were collected from freshly slaughtered healthy horses and either flash-frozen in liquid nitrogen or stored in RNAlater (Invitrogen). Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from blood of healthy horses by density-gradient centrifugation through Ficoll-Paque 1077 g/l (GE Healthcare). Stimulation of PBMC with human rIL-2 (Proleukin, Chiron) was performed with 200 U/ml for the indicated time, at a concentration of 5 × 105 cells/ml in RPMI-1640 supplemented with 10% fetal calf serum (FCS “Gold”; Bio&SELL), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, non-essential amino acids and 1 mM sodium pyruvate (GIBCO/Invitrogen). Magnetic cell separation (Miltenyi Biotec) was used for the isolation of lymphocyte subtypes. CD4 and CD8 positive cells were isolated with murine IgG1 primary mAb (compare “Cell transfection and flow cytometry” below) and anti-mouse IgG MicroBeads.
8
OTII CD4+ T Cell Adoptive Transfer and Influenza Infection
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OTII wild-type CD4+ T cells were purified by magnetic cell separation (Miltenyi Biotec) and labeled with cell trace violet (CTV) proliferation dye. A total of 106 cells were adoptively transferred by intravenous injection into Thy1 disparate C57BL/6 recipient mice. The next day, mice were anesthetized by isoflurane and inoculated intranasally with 250 50% tissue culture infective doses of PR8-OVA influenza virus. Expression of different markers was examined on day 4 or 5 after infection. In some experiments, a total of 106 OTII Tcf7-GFP/+ CD4+ T cells were adoptively transferred into recipient mice that were subsequently infected with PR8-OVA. Tcf7-GFP/+–high and –low cells were sorted on day 5 after infection, relabeled with a CPD (either CTV or eFluor 670), and restimulated to examine responses.
9
Activation and Differentiation of Mouse CD4+ T Cells
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Wild-type (WT) C57BL/6 and IRF4-knockout (KO) (14 (link)) littermate mice served as sources of CD4+ T cells. Both male and female mice were used at age 8–10 wk, and mice were housed in specific pathogen–free conditions. All experiments were conducted in accordance with National Institutes of Health and Columbia University Institutional Animal Care and Use Committee guidelines. All efforts were made to minimize animal suffering and the number of animals used. Naive CD4+ T cells were purified from spleens by magnetic cell separation (Miltenyi Biotec) and subsequently labeled with the cell proliferation dye (CPD) CellTrace Violet (Thermo Fisher). A total of 5 ×105 cells was seeded in 48-well tissue culture plates precoated with anti-CD3 (1 μg/ml) and anti-CD28(1 μg/ml) inIscove’s Modified EagleMedium (Corning) supplemented with 10% FBS, IL-2 (20 IU/ml), and IL-12 (10 ng/ ml). For experiments with retroviral expression of T-bet–GFP, cells were activated for ≥36 h before spinfection, as previously described (3 (link)). Following spinfection, cells were returned to the original culture media and cultured for an additional 48 h before flow cytometric analysis.
10
Isolation and Purification of PBMC Subsets
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Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Lymphocyte separation media, PAA, Pasching, Austria). Cell subsets were purified by magnetic cell separation according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch-Gladbach, Germany) and purity was confirmed by flow cytometry. Monocytes (average purity >95%) were isolated by positive selection using anti-CD14 microbeads. CD4+ T cells were isolated via negative depletion resulting in an average purity of >99%. In some experiments, CD4+ T cells were separated into CD45RO- and CD45RO+ cells using CD45RO beads (average purities 97% and 89%, respectively). CD4+CD25-T cells (average purity 85% CD25-) were obtained by CD4+CD25+ T cell depletion using CD25 MicroBeads.
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