Mice were transplanted as previously described.21 (link) In brief, recipient B6D2F1 mice were intravenously (i.v.) injected with 5×106 TCD-BM cells form WT B6 donors plus 1×106 T cells purified from either wild-type (WT) or MyD88−/− B6 donors on day 0 following lethal total body irradiation (TBI, 12Gy) delivered in two doses at 3-hour intervals. BALB/c recipients were transplanted with 5×106 TCD-BM cells from WT B6 donors plus 1×106 T cells purified from either WT or MyD88−/− B6 donors on day 0 following 6 Gy TBI. Isolation of T cells and TCD were performed using a Pan T cell Isolation kit II and anti-CD90-MicroBeads, respectively, and the autoMACS Pro system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Mice were housed in sterilized microisolator cages and received autoclaved hyperchlorinated drinking water for the first three weeks after BMT, and filtered water thereafter.
Anti cd90 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, Japan
Anti-CD90-MicroBeads are a type of magnetic separation reagent used for the isolation and enrichment of CD90-positive cells from biological samples. The product consists of superparamagnetic beads coated with antibodies specific to the CD90 antigen expressed on the surface of certain cell types.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 glutamax medium, by Thermo Fisher Scientific (2 mentions)
Automacs pro system, by Miltenyi Biotec (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Collagenase, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
T cell isolation kit, by Miltenyi Biotec (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
6 protocols using anti cd90 microbeads
1
Allogeneic Hematopoietic Cell Transplantation
2
In Vitro CD4+ T Cell Proliferation Assay
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For in vitro analysis, CD4+ cells from DO11.10 mice were labeled with 1 μM CFSE in PBS. The CD4-negative fraction was depleted from the remaining T cells using anti-CD90 microbeads (Miltenyi Biotec) and AutoMACS separation and used as antigen-presenting cells (APCs). APCs were irradiated (30 Gy for 28 min) and cultured with CD4+ cells in a final concentration of 2 × 106 cells/ml cRPMI [RPMI 1640 Glutamax medium (Gibco, Paisley, UK) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), 2-mercaptoethanol (1 mM), sodium pyruvate (1 mM), and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 25 mM)] with a ratio of 4:1 in 96-well round-bottom plates. pOVA constructs were added in various concentrations as indicated. Cells were incubated for 4 days at 37°C in a humidified 5% CO2 atmosphere. For analysis of proliferation, cells were stained and analyzed by flow cytometry by gating on pOVA-TCR+ CD4+ cells and calculating the geometrical mean of the fluorescence intensity (GMFI) of the CFSE signal. Fold CFSE dilution was determined [fold CFSE dilution: GMFI (PBS control)/GMFI (sample)].
3
Characterization and Sorting of CD90+ CSCs
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Unsorted CSCs were characterized by flow cytometry as described 11 . Cells were incubated with FITC‐, PE‐ or APC‐conjugated antibodies (from R&D Systems or BD Biosciences) against CD105, CD45 and CD90 for 60 min. Isotype‐identical antibodies served as negative controls. Quantitative analysis was performed using a CyAN flow cytometer with FlowJo software (Ashland, OR). Magnetically activated cell sorting (MACS) was performed using anti‐CD90 microbeads (Miltenyi Biotec) according to the manufacture's instructions. Such cell sorting fractionated unsorted CSCs into CD90‐positive CSCs (or CD90+ CSCs) and CD90‐negative CSCs (CD90− CSCs). To enable cell fate tracking, a cohort of cells were genetically labelled with green fluorescent protein (GFP). Ready‐to‐use pre‐made GFP lentiviral particles were purchased from GenTarget Inc (San Diego, USA). Briefly, CSCs were grown to 50–75% confluent in 24‐well plate. The culture media were removed and replaced with fresh and warm culture media. The lentiviral particles were thawed and added to the cell culture media at a MOI of 100:1. The cells were returned to the incubator; 72 hrs after transduction, the GFP positive cells were observed with a fluorescence microscopy.
4
OVA-specific CD4+ T cell proliferation
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For in vitro proliferation assays, CD4+ cells from DO11.10 mice were labeled with 1 µM CFSE (5`carboxyfluorescein succinimidyl ester). The CD4 negative fraction was depleted from the remaining T cells using anti-CD90 Microbeads (Miltenyi Biotec) and AutoMACS separation according to the manufacturer’s instructions and used as antigen-presenting cells (APCs). APCs were irradiated (30 Gray) and cultured with CFSE-labeled CD4+ cells in a final concentration of 2x106 cells/ml cRPMI (RPMI 1640 Glutamax medium, Gibco, supplemented with 10% FCS, penicillin 100 U/ml, streptomycin 100 µg/ml, 2-mercaptoethanol 1 mM, sodium pyruvate 1 mM and Hepes buffer 25 mM) 3:1 in 96-well round-bottom plates. Unconjugated pOVA or PEGylated peptides were added in concentrations as indicated. Cells were incubated for 4 days at 37°C in a humidified 5% CO2 atmosphere.
For determination of cell proliferation, cells were analyzed by gating on OVA-TCR+ CD4+ cells and calculating the geometrical mean of the fluorescence intensity (GMFI) of the CFSE signal. Fold CFSE dilution was determined as GMFI (PBS control)/GMFI (sample) and plotted on a log2-scale where every log step represents one division step.
For determination of cell proliferation, cells were analyzed by gating on OVA-TCR+ CD4+ cells and calculating the geometrical mean of the fluorescence intensity (GMFI) of the CFSE signal. Fold CFSE dilution was determined as GMFI (PBS control)/GMFI (sample) and plotted on a log2-scale where every log step represents one division step.
5
Isolation and Expansion of Tumor-Infiltrating Lymphocytes
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Tumor cell suspensions were prepared from solid tumors by enzymatic digestion in HBSS (Life Technologies) containing 1 mg/ml collagenase, 0.1 mg/ml DNAse I, and 2.5 U/ml of hyaluronidase (all from Sigma-Aldrich) with constant stirring for 2 hours at room temperature. The resulting suspension was passed through a 70-um cell strainer, washed once with HBSS and resuspended in PBS + 3% BSA to a concentration of 1 x 106 cells/ml for flow cytometric analysis. Cells were labeled with anti-CD90 microbeads according to the manufacturers’ instructions (Miltenyi Biotec) and purified using an autoMACS. After autoMACS purification, TIL were cultured for 5 days in the presence of IL-2 (3000 IU/ml). On day 5, TIL were collected for in vitro assays.
6
Allogeneic Bone Marrow Transplantation in Mice
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Mice. Female C57BL/6 (B6; H-2 b ), B6.SJL-Ptprc a (B6/SJL, H-2 b , CD45.1 + ), and BALB.B (H-2 b , CD45.2 + ) mice were purchased from Charles River Japan (Tokyo, Japan) or Jackson Laboratory (Bar Harbor, ME). All animal experiments were performed under the auspices of the Institutional Animal Care and Research Advisory Committee.
BMT. Mice were transplanted as previously described (14) . In brief, after 11Gy X-ray total body irradiation (TBI) delivered in 2 doses at 4 h intervals, BALB.B mice were intravenously injected with 5 × 10 6 splenic T cells and 5 × 10 6 T cell-depleted bone marrow (TCD-BM) cells from B6 or B6/SJL donors on day 0. Isolation of T cells and T-cell depletion were performed using the T cell isolation kit and anti-CD90-MicroBeads, respectively, and the AutoMACS (Miltenyi Biotec Japan, Tokyo, Japan). Mice were maintained in specific pathogen-free condition and received normal chow and autoclaved hyperchlorinated water (PH 4) for the first 3 weeks post-BMT and filtered water thereafter. Survival after BMT was monitored daily and the degree of clinical GVHD was assessed weekly by a scoring system as described previously (15) .
BMT. Mice were transplanted as previously described (14) . In brief, after 11Gy X-ray total body irradiation (TBI) delivered in 2 doses at 4 h intervals, BALB.B mice were intravenously injected with 5 × 10 6 splenic T cells and 5 × 10 6 T cell-depleted bone marrow (TCD-BM) cells from B6 or B6/SJL donors on day 0. Isolation of T cells and T-cell depletion were performed using the T cell isolation kit and anti-CD90-MicroBeads, respectively, and the AutoMACS (Miltenyi Biotec Japan, Tokyo, Japan). Mice were maintained in specific pathogen-free condition and received normal chow and autoclaved hyperchlorinated water (PH 4) for the first 3 weeks post-BMT and filtered water thereafter. Survival after BMT was monitored daily and the degree of clinical GVHD was assessed weekly by a scoring system as described previously (15) .
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