For generating human monocyte-derived DCs, peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects by Ficoll-Paque density gradient centrifugation. CD14+ monocytes were isolated by magnetic cell sorting kit (Miltenyi, 130-097-052) according to the manufacturer’s instructions. Purified CD14+ cells were cultured in 24-well plate in complete RPMI-1640 media and stimulated with 100 ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF) plus 100 ng/ml IL-4 for 5 days for induction of immature DCs. Subsequently, 100 ng/ml lipopolysaccharides (LPS) was added to induce DC maturation. Forty-eight hours later, the cells were used as human monocyte-derived DCs. CD11c+ cells were isolated from splenocytes by magnetic cell sorting kit (Miltenyi, 130-097,059) and used as mouse DCs.
Magnetic cell sorting kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The Magnetic cell sorting kit is a comprehensive system for the isolation and purification of specific cell populations from complex biological samples. The kit utilizes superparamagnetic microbeads coated with antibodies or ligands that bind to specific cell surface markers, allowing for the selective magnetic separation of target cells from the sample. The core function of this product is to enable efficient and reproducible cell sorting with high purity and recovery.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Amniomax 2, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cell strainer, by BD (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bz x800 fluorescent microscope, by Keyence (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mc38 cells, by Kerafast (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
P stat6, by Santa Cruz Biotechnology (1 mentions)
H3k4ac, by Santa Cruz Biotechnology (1 mentions)
Rna polymerase 2, by Santa Cruz Biotechnology (1 mentions)
Il 10, by BD (1 mentions)
Chip kit, by Merck Group (1 mentions)
P stat3, by Santa Cruz Biotechnology (1 mentions)
Hdac1, by Santa Cruz Biotechnology (1 mentions)
12 protocols using magnetic cell sorting kit
1
Generation of Human and Mouse Dendritic Cells
2
Isolation of Hepatic and Splenic Lymphocytes
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Intra-hepatic and splenic lymphocytes were isolated by perfusion of the liver with digestion buffer. After perfusion, the liver was homogenized and incubated at 37°C for 30 min. The digested liver/spleen cell suspension was centrifuged to remove hepatocytes and cell clumps. The supernatant was then centrifuged to obtain a pellet of cells depleted of hepatocytes to a final volume of 1 ml. Lymphocytes were then isolated from this cell suspension using 24% metrizamide gradient separation [31 (link)]. Cells were cultured or counted and stained for FACS analysis. For spleen cells; the spleens were meshed through cell strainers (40μm BD FALCON, USA), cells were sedimented by centrifugation at 800xg for 3 minutes, and then treated with lysis buffer at RT for 3 minutes, 9mL DMEM added and spinned as before [33 (link)]. Liver/spleen NK from lymphocytes were further isolated using a magnetic cell sorting kit (Miltenyi Biotec) according to manufacturer’s instructions.
3
Isolation of Human NK Cells
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Heparinized blood samples were obtained from 22 healthy volunteers and 72 patients with NAFLD. Mononuclear cells were isolated by centrifugation over a Ficoll‐hypaque density gradient (Pharmacia). After three washes in saline, cells were resuspended in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, as described.17 Human NK cells were isolated from mononuclear cells by using a magnetic cell sorting kit (Miltenyi Biotec) according to the manufacturer's instructions.
4
NAFLD Pathogenesis: NK Cell Profiling
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Example 1
Heparinized-blood samples of healthy volunteers and NAFLD patients and/or cirrhotic cases were obtained. Mononuclear cells were isolated by centrifugation over ficoll-hypaque (Pharmacia). After three washes in saline, cells were resuspended in medium of Roswell Park Memorial Institute 1640 with 10% FBS. Human NK cells were isolated from Peripheral blood lymphocytes (PBLs) using a magnetic cell sorting kit (Miltenyi Biotec) according to manufacturer's instructions. RNA was extracted, converted to cDNA and hybridized onto a spotted microarray.
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5
Isolation of hAFSCs from Amniotic Fluid
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The study was approved by the institutional review board of Keio University School of Medicine (no. 20140285) and informed consent was obtained from all the volunteer donors. Five‐milliliter amniotic fluid samples were obtained from 15‐ to 17‐week‐old pregnant women who underwent amniocentesis. CD117‐positive (CD117+) cells were isolated as hAFSCs, as described previously 17 , 18 , 19 , 20 . Briefly, within 2 hours, cells were centrifuged at 200g for 5 minutes. After removing the supernatant, the cell pellet was cultivated in growth medium comprising alpha Modified Eagle Minimum Essential Medium (α‐MEM; Invitrogen, Carlsbad, CA), 15% fetal bovine serum (FBS; Invitrogen), 1% l ‐glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 40% AmnioMax‐II (Life Technologies, Carlsbad, CA). After the cell population became subconfluent, these cells were stained with PE‐conjugated CD117 antibody and observed with a BZ‐X800 fluorescent microscope (KEYENCE, Osaka, Japan) to count the number of positive cells. CD117+ cells were isolated as hAFSCs using a Magnetic cell sorting kit (Miltenyi Biotec, Auburn, CA).
6
Adoptive Transfer of Naïve Immune Cells
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Naïve CD4+ and B220+ B cells were purified from the spleens of WT and Slamf[1 + 5 + 6]−/ − mice using a magnetic cell sorting kit (Miltenyi Biotec). Rag-1−/− recipient mice were injected with 5 × 106 CD4+ T cells and 10 × 106 B220+ B cells in 200 μl PBS. Mice were immunized with NP-OVA/CFA 7 days after adoptive cell transfer.
7
Isolation and Characterization of Mouse MSCs and T Cells
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MSCs were generated from mouse bone marrow. Bone marrow cells were harvested and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin (all from Invitrogen, Carlsbad, CA, USA). After 8 h, non‐adherent cells were removed and adherent cells were maintained with medium replenishment every 2–3 days. Using a panel of surface markers, MSCs were determined. Positivity in markers of hematopoietic stem cells and lineage cells was used to identify non‐MSCs. The stemness was defined by the capacity to differentiate into adipocytes, osteoblasts, and chondrocytes.
Splenocytes were harvested from mice mentioned above. CD4+ or CD8+ T cells were isolated using magnetic cell sorting kit (Miltenyi, Bergisch Gladbach, Germany). T cells were maintained in RPMI‐1640 medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 U mL−1 penicillin, 100 µg mL−1 streptomycin, 1 mm sodium pyruvate, and 55 µm 2‐mercaptoethanol.
Splenocytes were harvested from mice mentioned above. CD4+ or CD8+ T cells were isolated using magnetic cell sorting kit (Miltenyi, Bergisch Gladbach, Germany). T cells were maintained in RPMI‐1640 medium supplemented with 10% fetal bovine serum, 2 m
8
Isolation and Characterization of hAFSCs
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hAFSCs were isolated from amniotic fluid using our previously reported method [14 (link)]. Briefly, the collected amniotic fluid was centrifuged and the amniotic fluid cells were isolated. After the cells were cultured, CD117-positive cells were isolated using a magnetic cell sorting kit (Miltenyi Biotec, Auburn, CA, USA). According to the criteria defined by the International Society for Cellular Therapy for MSCs, the obtained cells were confirmed to be positive for mesenchymal markers and negative for hematopoietic markers by flow cytometry and to have the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Subsequently, hAFSCs were cultured in the growth medium, which was composed of α-modified Eagle minimum essential medium (αMEM; Invitrogen, Carlsbad, CA, USA), 15% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), 1% L-glutamine (Invitrogen, Carlsbad, CA, USA), 1% penicillin/ It consisting of streptomycin (Invitrogen, Carlsbad, CA, USA), and 40% AmnioMax-II (Life Technologies, Carlsbad, CA, USA), and used in the experiments.
9
Transcriptomic Analysis of NASH NK Cells
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Example 1
Heparinized-blood samples of healthy volunteers and NAFLD patients and/or cirrhotic cases were obtained. Mononuclear cells were isolated by centrifugation over ficoll-hypaque (Pharmacia). After three washes in saline, cells were resuspended in medium of Roswell Park Memorial Institute 1640 with 10% FBS. Human NK cells were isolated from Peripheral blood lymphocytes (PBLs) using a magnetic cell sorting kit (Miltenyi Biotec) according to manufacturer's instructions. RNA was extracted, converted to cDNA and hybridized onto a spotted microarray.
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10
Isolation and Culture of Murine MSCs and CD8+ T Cells
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MSCs were isolated from bone marrow of 6-week-old female C57/BL6 mouse tibia and femur according to the protocol previously described by our laboratory [57 (link)]. Ctrl-MSCs and IFNα-MSCs were constructed by lentivirus transfection as previously described [27 (link)]. MC38 cells were purchased from Kerafast (Boston, MA). B16F10 cells and MC38 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. All cell lines were tested negative for Mycoplasma contamination and authenticated with short tandem repeat assays. Splenic or intra-tumoral CD8+ T cells were isolated using magnetic cell sorting kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ T cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 55 μM 2-mercaptoethanol (Gibco, Grand Island, NY).
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