Mature dendritic cells were differentiated from blood monocytes as previously described (Bruger et al, 2020 (link)). Briefly, monocytes (isolated from mononuclear cells of non-cancerous patients as described above) were resuspended in RPMI-1640 complemented with 10% FCS (Sigma-Aldrich), 200 nM L-alanyl-L-glutamine dipeptide (35050-038, GlutaMAX; Gibco), 100 U/ml penicillin + 100 μg/ml streptomycin (P4333; Sigma-Aldrich), 100 ng/ml of clinical grade GM-CSF (NDC0024-5843-05, Leukine [Sargramostim]; Sanofi-Aventis), and 30 ng/ml (5 × 105 U/ml) IL-4 (in-house) (DC medium). Monocytes were seeded in a 12-well tissue culture plate at 106 cells/well, and incubated for 5 d at 37°C and 5% CO2. Dendritic cells were matured at Day 5 with 50 μg/ml Poly-IC (P1530; Sigma-Aldrich) and 10 ng/ml TNF-α (300-01A; PeproTech) in DC medium for another 2 d at 37°C and 5% CO2. Cells were harvested at Day 7 and checked by flow cytometry to confirm their phenotype (CD3−, CD19−, CD14−, CD11c+, CD1a+, CD83+) (see antibodies for flow cytometry). Mature DC were frozen at −80°C in 50% RPMI-1640, 40% FCS, and 10% DMSO for later use.
Streptomycin p4333
Manufactured by Merck Group
Sourced in United States
Streptomycin (P4333) is a laboratory reagent manufactured by Merck Group. It is an antibiotic agent commonly used in microbiology and molecular biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (5 mentions)
Tnf α 300 01a, by Thermo Fisher Scientific (1 mentions)
Leukine sargramostim, by Sanofi (1 mentions)
Poly ic p1530, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Low protein binding filter, by Merck Group (1 mentions)
Sodium palmitate p9767, by Merck Group (1 mentions)
A8806, by Merck Group (1 mentions)
L glutamine g7513, by Merck Group (1 mentions)
D glucose g8270, by Merck Group (1 mentions)
2 mercaptoethanol m3148, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Anti cd28 antibody, by BioLegend (1 mentions)
Anti ifn γ xmg1, by BioLegend (1 mentions)
Anti il 4 11b11, by BioLegend (1 mentions)
Tgfβ1 100 21c, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by BioLegend (1 mentions)
Il 12, by BioLegend (1 mentions)
7 protocols using streptomycin p4333
1
Monocyte-Derived Dendritic Cell Protocols
2
Characterizing Human Schlemm's Canal Cells
3
Metabolic Activation of Bone Marrow Macrophages
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Bone marrow cells were isolated from femurs of 3–6 months old male TG2 null mice and their wild type counterparts. Bone marrow macrophages (BMDMs) were differentiated in DMEM supplemented with 10% FBS (12106C), 2 mM L-glutamine (G7513), 1 mM Na-pyruvate (S8636), 50 μM 2-mercaptoethanol (M3148) and 100 U/ml penicillin/100 μg/ml streptomycin (P4333) all from Sigma-Aldrich and 10% L929 fibroblast conditioned media for 7 days. For metabolic activation, differentiated macrophages were treated with a combination of 30 mM D-glucose (G8270), 10 nM insulin (12643) and 0.4 mM sodium-palmitate (P9767) all from Sigma-Aldrich for 24 h (16). Sodium palmitate was prepared by diluting a 200 mM stock solution in 70% ethanol into 10% fatty acid-free, low-endotoxin BSA (Sigma Aldrich, A8806 adjusted to pH 7.4) to obtain a 5 mM palmitate-BSA stock solution that was filtered using a 0.22-μm low-protein binding filter (Millipore). BSA/ethanol was used in control treatments during the protocol. In some experiments during the 24 h metabolic activation BMDMs were treated with PP2 (Sigma Aldrich, 529573), a reversible ATP-competitive inhibitor of the Src family of protein tyrosine kinases, in 2 µM final concentration or 0.5 mg/ml RGD peptide (Cayman Chemical, 529573).
4
In Vitro T Cell Polarization Assay
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Purified naïve CD4+ T cells were cultured in 24-well (4 x 105 cells per well), 48-well (2 x 105 cells per well) or 96-well (1 x 105 cells per well) plates coated with 5 μg/mL anti-CD3ϵ antibody (145-2C11; Biolegend) in IMDM medium (12440-061; Invitrogen) containing 1 μg/mL anti-CD28 antibody (37.51; Biolegend), 10% FBS, 1x streptomycin-penicillin (containing 100 U/mL penicillin and 100 μg/mL streptomycin, P4333; Sigma), and 55 μM β-mercaptoethanol (20985-023; Invitrogen, Waltham, MA, USA). In addition, the following cytokines and antibodies were added in each polarizing condition: 20 ng/mL IL-2 (570402; Biolegend), 1 μg/mL anti-IFN-γ (XMG1.2; Biolegend), and 1 μg/mL anti-IL-4 (11B11; Biolegend) for Th0; 20 ng/mL IL-2, 20 ng/mL IL-12 (577002; Biolegend), and 1 μg/mL anti-IL-4 for Th1; 20 ng/mL IL-2, 100 ng/mL IL-4 (574306; Biolegend), and 1 μg/mL anti-IFN-γ for Th2; 20 ng/mL IL-6 (575706; Biolegend), and 3 ng/mL TGF-β1 (100-21C; PeproTech, Cranbury, NJ, USA) for Th17. Polarized cells were harvested for further analysis at the indicated time points.
5
Cell Culture Protocols for Leukemia and Vero Cells
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The human leukemia cell lines K562, TCCY-T315I and Ba/F3 were received from Prof. Yuko Sato (Tokyo, Japan). The Ba/F3 cells with T315I, Y253H and E279K were created as previous reported [20 (link)]. The African green monkey kidney (Vero) cell lineage (ATCC CCL-81™) was used in this study. Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, Ho Chi Minh City, Vietnam) and other cells were grown in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Sigma-Aldrich, Ho Chi Minh City, Vietnam) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (ThermoFisher Scientific, Ho Chi Minh City, Vietnam), 100 IU/mL penicillin, and 0.1 mg/mL streptomycin (P4333, Sigma-Aldrich, Ho Chi Minh City, Vietnam) in a humidified incubator of 5% CO2 at 37 °C.
6
Glioblastoma Primary Brain Tumor Cells
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We used previously established primary brain tumor initiating cell lines (BTIC)-10 and -12 derived from patients with IDH wild type glioblastoma as described15 (link),41 . The Department of Neuropathology, University of Regensburg (MJR), verified the patients’ diagnoses and WHO grade.
Tumor cells were maintained in RHB-A (Y40001, Takara), supplemented with 20 ng/ml of EGF (130097751), bFGF (130093842) (both Miltenyi Biotech), and 50 U (v/v) Penicillin/0.05% (v/v) Streptomycin (P4333) (Sigma-Aldrich) at 37 °C, 5% CO2, 95% humidity in a standard tissue culture incubator. Progenitor features of BTICs were verified by clonogenicity assays, flow cytometry (CD133, CD15, CD44, A2B5), immunocytochemistry (Nestin, Sox2, GFAP), and tumor take in an immunocompromised mouse model (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BTICs were lentivirally transduced to achieve stable expression of the nuclear fluorescent protein H2B Dendra2, as described61 (link).
The ethics board of the University of Regensburg, Germany, approved the use of human material for this study (No° 11-103-0182) and all patients gave written informed consent. All methods were performed in accordance with the relevant guidelines and regulations.
Tumor cells were maintained in RHB-A (Y40001, Takara), supplemented with 20 ng/ml of EGF (130097751), bFGF (130093842) (both Miltenyi Biotech), and 50 U (v/v) Penicillin/0.05% (v/v) Streptomycin (P4333) (Sigma-Aldrich) at 37 °C, 5% CO2, 95% humidity in a standard tissue culture incubator. Progenitor features of BTICs were verified by clonogenicity assays, flow cytometry (CD133, CD15, CD44, A2B5), immunocytochemistry (Nestin, Sox2, GFAP), and tumor take in an immunocompromised mouse model (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BTICs were lentivirally transduced to achieve stable expression of the nuclear fluorescent protein H2B Dendra2, as described61 (link).
The ethics board of the University of Regensburg, Germany, approved the use of human material for this study (No° 11-103-0182) and all patients gave written informed consent. All methods were performed in accordance with the relevant guidelines and regulations.
7
Isolation and Culture of Human NK Cells
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Preparation and culture of peripheral blood NK cells Polyclonal primary human NK cells were isolated by negative selection from lymphocyte-enriched buffy coat residues derived from anonymous healthy donors as previously described. 23 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat by centrifugation using Ficoll-Paque Plus according to the manufacturer's instructions (GE Healthcare). NK cell isolation was performed by negative immunomagnetic bead isolation (EasySep® Human NK Cell Enrichment Kit, Stemcell Technologies) according to the manufacturer's instructions. Primary human NK cells were cultured for 7-14 days in Iscove's modified Dulbecco's medium (IMDM, I3390, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% autologous human serum, 2 mM L-glutamine (G7513, Sigma-Aldrich, St. Louis, MO, USA), 100 U ml -1 penicillin-100 μg ml -1 streptomycin (P4333, Sigma-Aldrich, St. Louis, MO, USA), 1× non-essential amino acids (M7145, Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate (S8636, Sigma-Aldrich, St. Louis, MO, USA) and 200 U ml -1 human interleukin (IL)-2 (PeproTech, Rocky Hill, NJ, USA) before use in experiments.
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