PBMCs were isolated from buffy coats by Ficoll density gradient centrifugation (Biocoll Separation Solution, Merck KGaA, Darmstadt, Germany). CD14+ monocytes were isolated from total PBMCs by magnetic cell sorting using CD14 microbeads (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). PBMCs or CD14+ monocytes were seeded at a density of 1×106 or 1×105 per well in a 96-well plate, respectively, and cultured with 200 µL Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza, Köln, Germany) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich, St. Louis, MO, USA). Monocyte-derived macrophages were differentiated from CD14+ cells with 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Leukine® (Sargramostim), Sanofi, Paris, France), immature dendritic cells (iDCs) were differentiated from CD14+ cells with 50 ng/mL GM-CSF and 50 ng/mL interleukin (IL)-4 (Peprotech, Rocky Hill, NJ, USA). Mature DCs (mDCs) were generated by stimulating iDCs with 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) for 24 h.
Iscove s modified dulbecco s medium imdm
Manufactured by Lonza
Sourced in Germany, Switzerland, Belgium
Iscove's modified Dulbecco's medium (IMDM) is a cell culture medium commonly used for the in vitro cultivation of a variety of cell types, including hematopoietic and lymphoid cells. It is a modified version of Dulbecco's Modified Eagle's Medium (DMEM) and provides a defined, enriched environment for the growth and maintenance of these cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Lonza (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Lonza (3 mentions)
L glutamine, by Lonza (3 mentions)
Amphotericin b, by PAN Biotech (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions)
Interleukin il 4, by Thermo Fisher Scientific (1 mentions)
Biocoll separation solution, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Histopaque 1119, by Merck Group (1 mentions)
Hrgm csf, by Thermo Fisher Scientific (1 mentions)
Hrscf, by Thermo Fisher Scientific (1 mentions)
17 protocols using iscove s modified dulbecco s medium imdm
1
Isolation and Differentiation of Immune Cell Subsets
2
Isolation and Culture of CML Cells
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Blood or bone marrow samples were obtained from 76 CML patients (55 in CML-CP and 21 in CML-BP) with confirmed Philadelphia chromosome and BCR::ABL1 translocation and from 4 healthy volunteers. A peripheral blood mononuclear cells were isolated from CML patients or healthy blood donors using Histopaque 1077 and Histopaque 1119 (Sigma-Aldrich, Saint Louis, MO, USA), then progenitor CML CD34 + cells were selected using magnetic beads system EasySep CD34 + Positive Selection Kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s recommendation. CD34 + cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS) and growth factors: 2 ng/ml hrIL-3, 10 ng/ml hrGM-CSF and 2 ng/ml hrSCF (PeproTech, Cranbury, NJ, USA) at 37 °C in a humidified atmosphere of 5% CO2.
32D clone 3, interleukin (IL)-3-dependent cell line, and their BCR::ABL1-transformed counterparts were described before (Koptyra et al. 2006 (link)). The cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI, Lonza, Basel, Switzerland) with 10% FBS and 4 ng/ml mrIL-3 (PeproTech, Cranbury, NJ, USA) at 37 °C in a humidified atmosphere of 5% CO2.
32D clone 3, interleukin (IL)-3-dependent cell line, and their BCR::ABL1-transformed counterparts were described before (Koptyra et al. 2006 (link)). The cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI, Lonza, Basel, Switzerland) with 10% FBS and 4 ng/ml mrIL-3 (PeproTech, Cranbury, NJ, USA) at 37 °C in a humidified atmosphere of 5% CO2.
3
HLA-DQ/DR Molecules from EBV-transformed B Cells
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Lysates of Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell lines were used as a source for HLA-DQ/DR molecules: BOLETH (DQA1*03:01/DQB1*03:02), BSM (DQA1*03:01/DQB1*03:02), DUCAF (DRB1*03:01/DQA1*05:01/DQB1*02:01), JSM (DQA1*03:02/DQB1*03:01), and YOT (DRB1*04:05). Cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM) (Lonza Bioresearch, Basel, Switzerland) supplemented with 8 % heat-inactivated fetal calf serum (FCS) (Gibco; Life Technologies, Carlsbad, CA, USA), 1 % penicillin, streptomycin, and GlutaMAX (Invitrogen, Carlsbad, CA, USA).
4
Canine Mast Cell Activation Assay
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Ibrutinib was obtained from Selleck Chemicals (Houston, Texas), toceranib from Sigma‐Aldrich (St. Louis, Missouri), masitinib and midostaurin from LC laboratories (Woburn, Massachusetts). Stock solutions for all drugs were prepared by dissolving in dimethyl sulfoxide (DMSO) purchased from Sigma‐Aldrich. RPMI 1640 medium, Iscove's modified Dulbecco's medium (IMDM) and antibiotics (penicillin, streptomycin) were purchased from Lonza (Basel, Switzerland), amphotericin B from PAN‐Biotech (Aidenbach, Germany), fetal calf serum (FCS) from Gibco Life Technologies (Carlsbad, California), 3H‐thymidine from PerkinElmer (Waltham, Massachusetts), collagenase type 2 from Worthington (Lakewood, New Jersey) and trypan blue and 4′,6‐diamidino‐2‐phenylindole (DAPI) from Sigma‐Aldrich. DMSO was used as vehicle‐control in all experiments (corresponding to highest drug concentrations) and showed no effects on growth and activation of canine MCs (not shown).
5
Immune Cell Culture and Reagents
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Vitamin D was purchased from Selleck Chemicals (Houston, TX, USA) and dexamethasone and hydroxychloroquine from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of drugs were prepared by dissolving in dimethylsulfoxide (DMSO) (Merck, Darmstadt, Germany). RPMI 1640 medium, Iscove’s Modified Dulbecco’s Medium (IMDM), and antibiotics (penicillin + streptomycin) were purchased from Lonza (Verviers, Belgium), fetal calf serum (FCS) from Gibco, Life Technologies (Paisley, UK), alpha-thioglycerol from Sigma-Aldrich, and amphotericin B from PAN Biotech (Aidenbach, Germany). Recombinant human stem cell factor (SCF) was obtained from Peprotech (Cranbury, NJ, USA). Collagenase type II was purchased from Merk (Darmstadt, Germany) or Stemcell Technologies (Vancouver, BC, Canada). A specification of monoclonal antibodies (mAb) used in this study is shown in Supplementary Table S1 . Fc-blocking reagent was obtained from Militenyi Biotec (Bergisch Gladbach, Germany).
6
Culture and Maintenance of Cell Lines
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Human MDA-MB453 breast carcinoma and HEK 293T cells (both ATCC, Manassas, VA) were propagated in DMEM (Lonza, Cologne, Germany). Human Raji and WSU-NHL lymphoma, BV173 leukemia and C1R-neo B-cell lymphoblastoid cells (all ATCC) were cultured in RPMI 1640 medium (Lonza). Media were supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (Life Technologies, Darmstadt, Germany). MDA-MB453/CD19 breast carcinoma cells were generated by transduction with GALV pseudotyped retroviral vector encoding truncated human CD19 (kindly provided by Malcolm Brenner and Gianpietro Dotti, Baylor College of Medicine, Houston, TX) following standard protocols [30] . Primary human pre-B-ALL blasts were grown in Iscove's modified Dulbecco's medium (IMDM; Lonza) supplemented with 1 µg/mL bovine insulin (Sigma-Aldrich, Taufkirchen, Germany), 50 M b-mercaptoethanol, 200 µg/mL Fe 3+ -saturated human apo-transferrin (Invitrogen, Karlsruhe, Germany), 0.6% human serum albumin (Sanquin, Amsterdam, The Netherlands), 2.0 mM L-glutamine, and 20 µg/mL cholesterol [31] . Human NK-92 cells (kindly provided by NantKwest, Inc., Culver City, CA) were cultured in X-VIVO 10 (Lonza) containing 5% heat-inactivated human plasma (German Red Cross Blood Donation Service Baden-Württemberg -Hessen) and 100 IU/mL IL-2 (Proleukin; Novartis Pharma).
7
HLA-DP Peptide Identification in K562 Cells
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To investigate the peptides which are presented in the thirteen most frequently expressed HLA-DP molecules with more than 1% population frequency in the Netherlands, HLA class I and II–negative human immortalised myelogenous leukemia (K562) cell lines were transduced with individual HLA-DP alleles to obtain thirteen different K562 cell lines. The β-chain and the corresponding α-chain (Supplementary Table 1
8
Detailed Protocols for Prostate Cancer Cell Lines
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PC346C (p25) was generated in our lab as described previously (25 (link), 26 ) and maintained in prostate growth medium (PGM) consisting of DMEM-F12, supplemented with 2% FCS (both from Lonza, Breda, Netherlands), 1% insulin-transferrin-selenium (Gibco), 100 ng/ml fibronectin (Harbor Bio-Products, Tebu-bio, Netherlands), 20 μg/ml fetuin (ICN Biomedicals, Zoetermeer, Netherlands), 0.01% BSA, 10 ng/ml epidermal growth factor, 50 ng/ml cholera toxin, 0.1 mM phosphoethanolamine, 0.6 ng/ml triiodothyronine, 500 ng/ml dexamethasone (all from Sigma-Aldrich), 0.1 nM R1881 (Sigma), and penicillin/streptomycin antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin; Cambrex BioWhittaker). VCaP (p30) (20 ) (kindly provided by Dr. K.J. Pienta, Baltimore, MD, USA) and DuCaP (p52) (21 ) (kindly provided by Dr. J.A. Schalken, Nijmegen, Netherlands) were maintained in RPMI1640 (Lonza), supplemented with 10% FCS and penicillin/streptomycin antibiotics. LAPC4 (p49) (22 (link)) was kindly provided by Organon (Oss, Netherlands) and maintained in Iscove's Modified Dulbecco's Medium (IMDM) (Lonza), supplemented with 7.5% FCS and 10 nM R1881. Cell line authentication was performed by short tandem repeat analysis by the Promega PowerPlex 16 kit.
9
Sourcing and Culturing DLBCL Cell Lines
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Fresh-frozen biopsies of histologically confirmed DLBCL samples were identified in the pathology archive at Leiden University Medical Center (LUMC). The study was approved by the Scientific Review Committee of the LUMC Department of Hematology under an applicable waiver of consent by the LUMC Ethical Committee (B16.048).
DLBCL cell lines OCI-Ly3 (RRID:CVCL_8800), TMD8 (RRID:CVCL_A442), and U2932 (RRID:CVCL_1896) were obtained from the German Collection of Microorganisms and Cell Cultures. Phoenix cells (RRID:CVCL_H717) were cultured at 5% CO2 in Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza) supplemented with 10% fetal bovine serum (FBS; Bodinco), 3 mM L-glutamine (Lonza), 100 U/ml penicillin/streptomycin (Lonza). TKO cells were used, which are generated from the Oct cell line (RRID:CVCL_WN86) that originates from transformed murine pre-B cells from Slp65−/− mice. The TKO cell line has a Rag2 and Lambda5 KO. These cells were cultured at 7% CO2 in Iscove’s Basal Medium (Merck) supplied with 5% FBS (PAN BioTech), 3 mM L-glutamine, 100 U/ml penicillin/streptomycin, 50 mM 2-mercaptoethanol (Sigma-Aldrich; Merck), and IL-7–containing supernatant of mouse plasmacytoma cells J558L (RRID:CVCL_3949), stably transfected with a murine IL-7 expression vector (Meixlsperger et al., 2007 (link)).
DLBCL cell lines OCI-Ly3 (RRID:CVCL_8800), TMD8 (RRID:CVCL_A442), and U2932 (RRID:CVCL_1896) were obtained from the German Collection of Microorganisms and Cell Cultures. Phoenix cells (RRID:CVCL_H717) were cultured at 5% CO2 in Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza) supplemented with 10% fetal bovine serum (FBS; Bodinco), 3 mM L-glutamine (Lonza), 100 U/ml penicillin/streptomycin (Lonza). TKO cells were used, which are generated from the Oct cell line (RRID:CVCL_WN86) that originates from transformed murine pre-B cells from Slp65−/− mice. The TKO cell line has a Rag2 and Lambda5 KO. These cells were cultured at 7% CO2 in Iscove’s Basal Medium (Merck) supplied with 5% FBS (PAN BioTech), 3 mM L-glutamine, 100 U/ml penicillin/streptomycin, 50 mM 2-mercaptoethanol (Sigma-Aldrich; Merck), and IL-7–containing supernatant of mouse plasmacytoma cells J558L (RRID:CVCL_3949), stably transfected with a murine IL-7 expression vector (Meixlsperger et al., 2007 (link)).
10
Cell Culture Conditions for Various Cell Lines
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C1R HLA-A2 cells, Jurkat cells, and J.RT3-T3.5 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine (Lonza). The generation of C1R HLA-A2 cells was described previously (15 (link)). JE6.1 Jurkat cells, K562 A2 cells, K562 HLA-A2+CD20 cells, Raji HLA-A2 cells, Raji HLA-A2+CTAG1 cells, and U266 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1.5% L-glutamine (Lonza). ALL CM cells were cultured in IMDM containing serum-free supplement and 1% penicillin/streptomycin as described previously (48 (link)). UM3 cells were cultured in IMDM supplemented with 20% FBS, 1% penicillin/streptomycin, 1.5% L-glutamine, and 10 ng/ml IL-6 (Lonza). HEK 293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, and 1 mM HEPES (Lonza). Phoenix Ampho cells were cultured in IMDM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine (Lonza).
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