Blood, spleen, peritoneal leukocytes, and mesenteric lymph nodes were harvested from C57BL/6, BALB/c, and LDLR−/− mice. Blood, spleen, bone marrow, and nondraining lymph nodes were harvested before (pt) and 1 day after induction of HLI in LDLR−/−/CCCR7−/− and LDLR−/− mice. Draining lymph nodes were dissected from the ipsilateral inguinal region, nondraining form the contralateral iguinal region. Total circulating leukocytes were measured using a KX‐21N Hematology Analyzer (Sysmex). Tissues were minced through a 40‐μm cell strainer (BD Biosciences) to obtain single‐cell suspensions, which were resuspended in Iscove's Modified Dulbecco's Medium (Lonza) with 2% FCS. For DC‐specific cell surface staining, the spleen and lymph nodes were first perfused with collagenase (1 mg/mL) and DNase (0.02 mg/mL) for 10 minutes and minced. Erythrocytes were lysed and samples for intracellular staining were permeabilized. Fluorochrome‐conjugated monoclonal antibodies specific for CD3, CD4, CD8, CD11c, CD11b, CD19, CD25, CD86, CD115, FoxP3, Ly6C, Ly6G, B220, DX5, MHCII, CCR7, CCR2, F4/80, CD62L, IL12, IL10, NK1.1, and Tbet were used.
Iscove s modified dulbecco s medium
Manufactured by Lonza
Sourced in Belgium, Switzerland, United States
Iscove's modified Dulbecco's medium is a cell culture medium developed for the in vitro cultivation of a variety of mammalian cell types. It is a modified version of Dulbecco's Modified Eagle's Medium (DMEM) and is designed to support the growth and maintenance of cells.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Lonza (5 mentions)
L glutamine, by Lonza (3 mentions)
Rpmi 1640 medium, by Lonza (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fixation and permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Anti foxp3 mab, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Dulbecco s modified eagle s medium, by Lonza (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Methocult m3434, by STEMCELL (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
40 μm cell strainer, by BD (1 mentions)
Kx 21n hematology analyzer, by Sysmex (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
30 protocols using iscove s modified dulbecco s medium
1
Immune Cell Characterization in Ischemic Mice
2
Treg Cell Analysis in Blood Samples
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PB mononuclear cells (PBMC) were isolated by density gradient centrifugation from heparinized venous blood. PBMC were stained for Treg cells with anti-CD4, anti-CD25, and anti-CD127 monoclonal antibodies (mAb) (Biolegend, San Diego, CA) on the cell surface. For detection of the transcriptional factor FoxP3, cells were fixed with fixation and permeabilization buffers (eBioscience, San Diego, CA) and were then stained with anti-FoxP3 mAb (eBioscience). The expression of IL-17 and IFN-γ was analyzed by intracellular cytokine staining.8 (link),20 (link) PBMC were cultured in Iscove's modified Dulbecco's medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and stimulated for 5 hours with Phorbol 12-myristate 13-acetate PMA (50 ng/mL) and ionomycin (500 ng/mL) in the presence of brefeldin A (10 μg/mL, Sigma-Aldrich, St. Louis, MO). Cells were first stained for the surface antigen CD4 (Biolegend) and then fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin, followed by intracellular staining with anti-IL-17 and anti-IFN-γ mAbs (Biolegend). Stained PBMC were acquired on a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Ashland, OR).
3
Esophageal Adenocarcinoma Patient-Derived Xenograft
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Tumor material of patients diagnosed with EAC in the Academic Medical Center (Amsterdam, the Netherlands) was collected as described earlier and approved by the institute’s ethical committee and performed according to the guidelines of the Helsinki Convention (MEC 01/288#08.17.1042) (Damhofer et al., 2015 (link)). Patient material was expanded by subcutaneous xenograft in immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, which were bred and maintained at the local animal facility according to the legislation and ethical approval by the animal experiment ethical committee (LEX100780 and LEX102774). Primary cultures were established as described earlier (Damhofer et al., 2015 (link)). The primary culture used for these studies is AMC-EAC-007B (007B), a pretreatment biopsy, and cells were cultured according to standard culture procedures in Iscove’s Modified Dulbecco’s Medium supplemented with 8% fetal calf serum, L-glutamine (2 mmol/l), penicillin (100 units/ml), and streptomycin (500 µg/ml; Lonza, Basel, Switzerland).
4
Isolation and Expansion of Umbilical Cord Blood Cells
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Human UCB was obtained from donors after informed consent according to the Declaration of Helsinki. The experimental usage of UCB samples was approved by the local ethics commission. Mononuclear cells (MNCs) were isolated from individual sources by Ficoll (Biocoll Separating Solution, Biochrom AG) density gradient centrifugation and highly enriched by magnetic cell separation as described previously (Giebel et al., 2004 (link)). If not used immediately, cells were cultured in a humidified atmosphere at 37°C and 5% CO2 at densities of 0.5–1 × 105 cells/ml in Iscove’s modified Dulbecco’s medium (Lonza) supplemented with 20% fetal bovine serum (Biochrom), 100 U/ml penicillin, and 100 U/ml streptomycin (Life Technologies) and with early-acting cytokines (FLT3L, stem cell factor, and thrombopoietin, each at 10 ng/ml final concentration [all PeproTech]).
5
HUVEC and HEK293T Cell Culture
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HUVECs were purchased from Lonza and cultured on fibronectin-coated dishes at 37°C in 5% CO2 atmosphere. EGM2 medium supplemented with SingleQuots (Lonza) was used for culturing HUVECs. Cells were used for experiments until passage 5. HEK293T cells were purchased from ATCC and cultured in Iscove’s modified Dulbecco’s medium (Lonza) supplemented with 10% FCS and l- glutamine. Cells were used until passage 35 for production of lentiviral particles, native coimmunoprecipitation and immunoprecipitation of ubiquitinated proteins under denaturing conditions.
6
Isolation and Culture of CLL Cells
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Subjects were CLL patients (n=34) and healthy donors (n=3) seen at the ‘Centre Hospitalier de Luxembourg'. The clinical features of patients are summarized in Supplementary Table 1 . The experiments were approved by the ‘Comité National d'Ethique de Recherche' and all subjects signed an informed consent according to the Declaration of Helsinki.
Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (MP Biomedicals, Eindhoven, The Netherlands). PBMCs and Mec-1 cells were cultured in Iscove's modified Dulbecco's medium (Lonza, Verviers, Belgium), MCF-7 cells, JVM-2 and JVM-3 cells in RPMI-1640 medium (Lonza) and HeLa cells in Dulbecco's modified Eagle's medium, all containing 10% fetal bovine serum. The cells were incubated at 37 °C in an atmosphere of 5% CO2.
Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (MP Biomedicals, Eindhoven, The Netherlands). PBMCs and Mec-1 cells were cultured in Iscove's modified Dulbecco's medium (Lonza, Verviers, Belgium), MCF-7 cells, JVM-2 and JVM-3 cells in RPMI-1640 medium (Lonza) and HeLa cells in Dulbecco's modified Eagle's medium, all containing 10% fetal bovine serum. The cells were incubated at 37 °C in an atmosphere of 5% CO2.
7
Isolation and Cryopreservation of PBMC for CAR-T Cytotoxicity Assays
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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll Paque Plus (Cytiva, USA) from whole blood samples obtained from healthy donors. The PBMCs were cultured in Xvivo 15 medium (Lonza, Belgium), and cryopreserved in fetal bovine serum (FBS) containing 10% dimethyl sulfoxide. CD19-expressing luciferase-tagged K562 cells (Shanghai Genechem Co., Ltd.) were cultured in Iscove’s Modified Dulbecco’s Medium (Lonza, Belgium) supplemented with 10% FBS and used as target cells for the assessment of CAR-T cell cytotoxicity. Raji cells were cultured in RPMI 1640 medium supplemented with 10% FBS and used for the assessment of CAR-T cell efficacy in vivo.
8
Mesenchymal Stem Cells Modulate B Cell Activation
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Splenocytes were isolated from spleens of deceased organ donors (Biobank Erasmus MC protocol No. MEC-2012-022) by Ficoll density gradient separation (GE Healthcare, Uppsala, Sweden). Quiescent B cells were obtained by negative selection using anti-CD43 magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). B cells were cultured for 7 days in Iscove's modified Dulbecco's medium (Lonza) supplemented with 10% HI FBS and stimulated with F(ab)2 anti-IgM ( Jackson, ImmunoResearch laboratories, Inc., West Grove. PA), IL-2 (10 3 IU, Proleukin; Prometheus Laboratories, Inc., San Diego, CA), and 5 mg/mL anti-CD40 agonistic monoclonal antibody (Bioceros, Utrecht, The Netherlands). Inactivated and control MSC were added to the culture at day 0 in a MSC:B cell ratio of 1:5. IL-10 levels in the supernatant were measured using a human IL-10 ELISA kit (U-Cytech, Utrecht, The Netherlands) according to the manufacturer's protocol.
9
Culturing Various Cell Lines
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Human lung adenocarcinoma A549 cells were cultured in Ham-F12 (Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 100 U/mL penicillin-streptomycin-glutamine (Pen-Strep-Glut, Lonza) mixture. MDCK cells were cultured in Eagle’s minimal essential medium (EMEM; Lonza) plus 10% FBS, 1% Hepes 1 M, 1% sodium bicarbonate (Lonza), 1× non-essential amino acid solution, and 100 U/mL Pen-Strep-Glut mixture. A subclone 118 of Vero-WHO cells (Vero-118 (36 (link)) was cultured in Iscove’s modified Dulbecco’s medium (Lonza) supplemented with 10% FBS, and 100 U/mL Pen-Strep-Glut mixture. Jurkat cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco), supplemented with 10% FBS, 100 U/mL Pen-Strep, and 1% Hepes 1 M (all Gibco). All cells were cultured at 37°C and 5% CO2. Monocytic THP-1 cells were cultured in RPMI 1640 (Gibco), supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol (Gibco), and 100 U/mL Pen-Strep (Sigma-Aldrich).
10
Cell Line Authentication and Culture
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We used four human SCC cell lines, CaSki and SiHa, from uterine cervix and A431, from skin, along with the TPT-resistant subline (A431/TPT) selected in our lab [24 (link)], two human Ewing's sarcoma family of tumor cell lines SK-N-MC (Askin's tumor) and TC71 (Ewing's sarcoma) and the human embryonal rhabdomyosarcoma cell line RD. All SCC, SK-N-MC and RD cell lines were cultured in RPMI-1640 medium (Lonza, Verviers, Belgium), whereas the TC71 cell line was maintained in Iscove's modified Dulbecco's Medium (Lonza). All cell lines were grown in medium supplemented with 10% FBS and maintained in a humidified incubator with 5% CO2 at 37°C. The A431, CaSki and SiHa cell lines were obtained from American Type Culture Collection (Manassas, VA). The SK-N-MC cell line was kindly provided by R. Maggi (University of Milan, Italy), the RD cell line by A. Rosolen (University of Padua, Italy), and the TC71 cell line by M.C. Manara (Rizzoli Institute, Bologna, Italy). Cell lines were authenticated by single tandem repeat analysis by the AmpFISTR Identifiler PCR amplification kit (Applied Biosystems, Foster City, CA).
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