After enrichment with stem cell medium, the LFs and BM MSCs were treated with 0.25% Trypsin-EDTA, resuspended in PBS at a concentration of 5×104 cells/μl and incubated with 1 μl of anti-SSEA4 antibody (R&D Systems Inc., Minneapolis, http://www.rndsystems.com ) for 30 minutes at 4°C (Fig. 1 ). The cells were then washed twice with PBS to remove unbound antibody and the cell suspension was incubated with 10 μl of magnetic beads tagged with goat anti-mouse IgG (Miltenyi Biotech, Auburn, CA, https://www.miltenyibiotec.com ) for 20 minutes at 4°C. The cell suspension was then subjected to magnetic-activated cell sorting columns (Miltenyi Biotech) for separation. The SSEA4+ and SSEA4− fractions of LFs and BM MSCs were replated every 5–6 days with KnockOut ESC/iPSC culture medium at a dilution of 1:3, under similar culture conditions. At each passage, population-doubling time was measured using this formula: number of cell doublings (NCD) = log10(y/x)/log102, where y is the final density of the cells and x is the initial seeding density of the cells.
Goat anti mouse igg
Manufactured by Miltenyi Biotec
Sourced in United States
Goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) molecules. It is used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and quantify the presence of mouse IgG in samples.
Automatically generated - may contain errors
3 protocols using goat anti mouse igg
1
Isolation and Expansion of SSEA4+ Stem Cells
2
Isolation and Purification of Plasmacytoid Dendritic Cells
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The study protocol was approved by the Institutional Ethics Committee of 306th Hospital of PLA. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll centrifugation. Monocytes, NK cells, T cells and B cells were depleted using a mixture of anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20, and anti-CD56 mAbs (Biolegend, San Diego, CA, USA) with magnetic beads coated with goat anti–mouse IgG (MiltenyiBiotec, San Diego, CA, USA). The lineage (CD3, CD14, CD16, CD19, CD20) depleted PBMC were stained with HLA-DR (APC-Cy7), CD123 (BV421), CD11c (APC), and HLA-DR+CD123+CD11c−Lin−pDCs were sorted with BD FACSAriaIII (BD Biosciences, Franklin Lake, NJ, USA), and the purity of pDCs was over 99%.
3
Porcine Monocyte-Derived Dendritic Cell Isolation
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Blood samples were taken from two minipigs and samples were processed separately. The monocyte-derived dendritic cells were isolated and cultured as described in Bimczok et al. (2007 (link)). The PBMCs (peripheral blood mononuclear cells) were stained with a mouse anti-SWC-3a antibody (CD172a; clone 74-12-15; VMRD, Pullman, WA) and then separated with magnetic microbeads (goat anti-mouse IgG; Miltenyi Biotec). Live/dead assay was examined with propidium iodide (10 µL/200 µL; stock solution: 1 mg/mL; Sigma, Germany). Monocyte-derived dendritic cells were generated in DMEM supplemented with 10% porcine serum (PAN-Biotech, Germany). A total of 1 × 106 cells/mL/well were seeded in 12-well plates and incubated at the porcine body temperature of 39 °C. They were stimulated with IL-4 (50 ng/mL) and GM-CSF (150 ng/mL, both Bioscience) for 7 days and then used for the subsequent in vitro experiments. On day 7, cells were centrifuged (350 × g; RT; 5 min) and detached with a pipette using cold medium. In the next step, cells were centrifuged at 350 × g and seeded for the experiments in DMEM supplemented with 10% porcine serum without IL-4 and GM-CSF.
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