Cell Lines RAW 264.7 cells (Merck, USA) were cultured in DMEM complete media (DMEM (Cellclone and Lonza, India) containing 10% fetal bovine serum (FBS, Thermofisher, USA) and antibiotics). Cells were passaged when the flasks were about 80% confluent. The HL-60 cells (ATCC, USA) were cultured in IMDM complete media (IMDM (Merck) containing 20% FBS and antibiotics). Cells were passaged when the cells were at a concentration of 1 million cells in 1 ml.
Iscove modified dulbecco media (imdm)
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Italy, Switzerland, Sweden
IMDM is a powdered cell culture medium developed by Iscove and Melchers. It is designed to support the growth and maintenance of a variety of cell types, including lymphoid, myeloid, and other hematopoietic cells. IMDM provides a balanced formulation of amino acids, vitamins, inorganic salts, and other essential components required for cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (23 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
L glutamine, by Thermo Fisher Scientific (17 mentions)
Fetal bovine serum (fbs), by Merck Group (16 mentions)
Streptomycin, by Thermo Fisher Scientific (16 mentions)
Penicillin, by Thermo Fisher Scientific (15 mentions)
Glutamax, by Thermo Fisher Scientific (14 mentions)
Streptomycin, by Merck Group (13 mentions)
Penicillin, by Merck Group (13 mentions)
L glutamine, by Merck Group (13 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (11 mentions)
Rpmi 1640, by Merck Group (9 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (9 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (7 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
β mercaptoethanol, by Merck Group (5 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (4 mentions)
138 protocols using iscove modified dulbecco media (imdm)
1
Cell Culture Protocols for RAW 264.7 and HL-60 Cells
2
Cell Culture Protocols for RAW 264.7 and HL-60 Cells
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Cell Lines RAW 264.7 cells (Merck, USA) were cultured in DMEM complete media (DMEM (Cellclone and Lonza, India) containing 10% fetal bovine serum (FBS, Thermofisher, USA) and antibiotics). Cells were passaged when the flasks were about 80% confluent. The HL-60 cells (ATCC, USA) were cultured in IMDM complete media (IMDM (Merck) containing 20% FBS and antibiotics). Cells were passaged when the cells were at a concentration of 1 million cells in 1 ml.
3
Cell Culture Optimization for Apoptosis Assays
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Cell densities were optimized for each individual assay and corresponding sampling time points, and peptide doses were adjusted to cell density to obtain the same number of annexin-positive cells in all experiments. These data are listed in Supplementary Table S1 , also listing number of independent cultures analysed (n), number of repeated experiments and time of harvest.
JJN3 [3 (link)] and RPMI 8226 (ATCC, Manassas, Virginia, USA, CCL-155) (MM), HL60 (ATCC CCL-240) and NB4 (Kind gift from professor Stein Døskeland, University of Bergen, Norway) (AML), and DU145 (ATCC HBT-81, prostate cancer) cells were cultured in RPMI 1640 (Sigma-Aldrich), MC/CAR (ATTC CRL-8083) (MM) were cultured in IMDM (Sigma-Aldrich or Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and HEK293 (ATCC CRL-1573, embryonic kidney) were cultured in DMEM high glucose (Sigma-Aldrich,), all supplemented with 2 mM glutamine (Biochrom, Berlin, Germany), 100 µg/mL gentamicin (Sigma-Aldrich or Gibco), 2.5 µg/mL amphotericin (Sigma-Aldrich), and fetal bovine serum (FBS)(Sigma-Aldrich); 10% in DMEM high glucose and RPMI 1640, and 20% in IMDM. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
JJN3 [3 (link)] and RPMI 8226 (ATCC, Manassas, Virginia, USA, CCL-155) (MM), HL60 (ATCC CCL-240) and NB4 (Kind gift from professor Stein Døskeland, University of Bergen, Norway) (AML), and DU145 (ATCC HBT-81, prostate cancer) cells were cultured in RPMI 1640 (Sigma-Aldrich), MC/CAR (ATTC CRL-8083) (MM) were cultured in IMDM (Sigma-Aldrich or Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and HEK293 (ATCC CRL-1573, embryonic kidney) were cultured in DMEM high glucose (Sigma-Aldrich,), all supplemented with 2 mM glutamine (Biochrom, Berlin, Germany), 100 µg/mL gentamicin (Sigma-Aldrich or Gibco), 2.5 µg/mL amphotericin (Sigma-Aldrich), and fetal bovine serum (FBS)(Sigma-Aldrich); 10% in DMEM high glucose and RPMI 1640, and 20% in IMDM. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2.
4
Generation of Bone Marrow-Derived Immune Cells
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Bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived myeloid cells (BMMCs) were generated using femurs and tibias of females C57BL/6 WT and Ifnar1−/− mice aged of 8 to 12 weeks. Bones were carefully collected in sterile PBS. After washing bones in alcohol and Iscove’s medium (IMDM, Sigma-Aldrich) baths, extremities of bones were cut and flushed using a 26 G needle. After red blood cell lysis with ACK buffer, cells were cultured in IMDM supplemented with 10% of FCS + 2 mM l -Glutamine + 100 UI/mL Penicillin/Streptomycin + 50 µM 2-mercaptoethanol (Sigma-Aldrich) (referred herein as complete IMDM medium) at 0.5 × 106/mL and treated with 10 ng/mL of GM-CSF and IL-4 for BMDCs and 50 ng/mL of M-CSF for BMMCs (all from Peprotech). Cells were split at day 3 and used at day 7 or 8.
5
Treg Differentiation and Suppression Assay
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Unless otherwise noted, Tregs were cultured in RPMI-1640 medium (plus β-mercaptoethanol) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin, 50 ng/ml IL-2 and anti-CD3/CD28 coated Dynabeads (Thermo Fisher) at 1:1 ratio (cells: beads). For glucose challenge experiments, glucose-free RPMI-1640 medium (Thermo Fisher, 1187020) were used. 2-DG was purchased from Sigma (D8375). For in vitro iTreg differentiation, 24-well plates were pre-coated with 40 μg/ml anti-hamster antibody (MP Biomedical). CD4+CD25−CD44−CD62L+ naive T cells were sorted and cultured for 3 days in IMDM (Sigma-Aldrich) supplemented with 0.25 μg/ml anti-CD3 (145-2C11), 1 μg/ml anti-CD28 (37.51), 2 μg/ml anti-IL-4 (11B11), 2 αg/ml anti-IFN-γ (XMG1.2) and 5 ng/ml TGF-β. For in vitro Treg suppression assay, splenocytes from CD45.1+ mice were labeled with Cell Trace Violet (Thermo Fisher) and cultured with or without various ratio of sorted splenic Tregs from control or Tfamfl/flFoxp3Yfp-Cre mice. Cells were cultured for 3d with soluble 1 μg/ml anti-CD3/CD28. The division of CD45.1+CD4+ T cells were assessed by the dilution of Cell Trace Violet.
6
Cell line authentication and cultivation
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HEK293T (CVCL_0063) cells, squamous cell lung carcinoma HARA (CVCL_2914) and LK-2 (CVCL_1377) cells, and colorectal cancer SW620 (CVCL_0547) cells were obtained from, verified as mycoplasma free, and validated by DNA fingerprinting by the Cell Services facility at The Francis Crick Institute. HARA and LK-2 cells were originally sourced from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (JCRB1080.0 and JCRB0829, respectively) and were deposited with the Cell Services Facility at The Francis Crick Institute. HARA cells were grown in RPMI 1640 Medium (Thermo Fisher Scientific) with 10% fetal bovine serum, and unless otherwise indicated, other cell lines were grown in IMDM (Sigma-Aldrich) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific). Media were further supplemented with l -glutamine (2 mmol/L, Thermo Fisher Scientific), penicillin (100 U/mL, Thermo Fisher Scientific), and streptomycin (0.1 mg/mL, Thermo Fisher Scientific).
7
Immortalized Megakaryocyte Cell Culture
8
Culturing Human Melanoma Cell Line
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LG2-MEL-220 (Mel220), a human PMEL-deficient melanoma cell line50 (link), was grown in IMDM (Sigma)/10% FCS (HyClone) containing non-essential amino acids (Gibco), GlutaMax (Gibco) and penicillin/streptomycin (Gibco). PMEL transfectants were grown in medium additionally containing 2 mg/ml G418 (Gibco).
9
Culturing Multiple Myeloma Cell Lines
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RPMI8226 were kindly provided by Dr Roberto Piva (Department of Pathology and CeRMS, University of Turin). OPM2 was purchased from DSMZ (Braunschweig, Germany). All MM cell lines were cultured in IMDM (Sigma-Aldrich Corp. St. Luis, MO, USA) containing 10% fetal bovine serum, 2mM L-glutamine (Gibco-Invitrogen, Grand Island, NY), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco).
10
Isolation and Morphological Analysis of Murine MDSCs
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Bone marrow cells were extracted from the femur and tibia of Balb/c mice, after which 10 × 106 bone marrow cells were cultured in 75% CM and 25% Iscove's Modified Dulbecco's medium (IMDM, Sigma-Aldrich) supplemented with 10% fetal clone I (FCI, GE Health Care Life Sciences, Hyclone Laboratories, Utah, USA), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich) for 6 days. Cell viability and cell numbers were evaluated by trypan blue staining (Sigma-Aldrich). To evaluate the morphology of the cells, 5 × 105 sorted MDSC were fixed on glass slides using the cytospin technique and were centrifuged at a speed of 1000 rpm for 5 minutes. Cytospin slides, filter cards, sample chambers, and metal clips were all obtained from Thermo scientific (Massachusetts, USA). Cytospins were air dried for 2 hours and afterwards stained with hematoxylin and eosin.
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