Bone-disassociated stromal cells (5,000) from CD45.2 mice and CD45.1+ LT-HSCs (1,000) were cultured in IMDM medium (Gibco) containing 10% FBS, 1 ng ml−1 KITL (Peprotech) and 1 ng ml−1 TPO (Peprotech) for 2 days. Harvested CD45.1+ HSCs were either sorted individually into 96-well U bottom plates for the single-cell CFU assay33 (link) or transplanted into lethally irradiated (1,100 rads) CD45.2 recipient mice (250 cells/mouse initial seeding HSC number) together with 200,000 CD45.2+ helper marrow cells. For the CFU assay the single cells were cultured in IMDM medium (Gibco) containing 10% FBS, 10ng ml−1 of the following cytokines (Peprotech) IL-1a, IL-3, IL-5, IL7, IL-9, IL-10, IL-11, GM-CSF, TPO, EPO, SCF and Flt3, colonies were counted 7 days after seeding. For the transplantation assay. For the transplantation assay, peripheral blood was drawn at the indicated time points and the contribution of donor-derived cells was analysed monthly by flow cytometry.
Imdm medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, China, Austria, Switzerland, France
IMDM medium is a cell culture medium formulation commonly used to support the growth and maintenance of various cell lines in laboratory settings. It provides a balanced nutrient composition to sustain cellular metabolism and proliferation. The core function of IMDM medium is to serve as a standardized, chemically-defined culture environment for in vitro cell studies.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (49 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (40 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (37 mentions)
Penicillin, by Thermo Fisher Scientific (23 mentions)
L glutamine, by Thermo Fisher Scientific (22 mentions)
Streptomycin, by Thermo Fisher Scientific (21 mentions)
Dmem medium, by Thermo Fisher Scientific (18 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (13 mentions)
Penicillin, by Merck Group (10 mentions)
Horse serum, by Thermo Fisher Scientific (9 mentions)
Glutamax, by Thermo Fisher Scientific (9 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (8 mentions)
β mercaptoethanol, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Merck Group (7 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Merck Group (6 mentions)
Rpmi medium, by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Merck Group (5 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (5 mentions)
Pen strep, by Thermo Fisher Scientific (5 mentions)
236 protocols using imdm medium
1
Hematopoietic Stem Cell Assays
2
Megakaryocyte Culture and Modulation
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Meg01 cell line (Daixuan, Shanghai, China) was incubated in an IMDM medium (Gibco, New York, USA) with 1% penicillin/streptomycin (Gibco, New York, USA) and 20% heat-inactivated fetal bovine serum (Gibco, New York, USA) for the study of megakaryocytes as previous studies in ITP [23 (link), 24 (link)]. Based on experimental grouping, PMA-induced Meg01 cells were treated with the selective COX-2 inhibitor celecoxib (Abmole, Houston, TX, USA) at different concentrations (0, 10, 20 and 40 μM) [25 (link), 26 (link)] or firocoxib at a concentration of 100 ng/ml in for three days, and then collected for the following experiments.
For the culture of mice bone marrow-derived megakaryocytes, a single cell suspension of bone marrow from wild C57BL/6 mice was collected and cultured in IMDM medium (Gibco, New York, USA) containing 1% penicillin/streptomycin (Gibco, New York, USA) and 20% heat-inactivated fetal bovine serum (Gibco, New York, USA) in the presence of 100 ng/ml recombinant murine thrombopoietin (rmTPO) and 100 ng/ml murine stem cell factor (SCF) for 7 days with or without 10 μM celecoxib. Megakaryocytes were purified and enriched by a 1.5%/3.0% discontinuous bovine serum albumin (BSA) gradient [27 (link)] and prepared for the following experiments.
For the culture of mice bone marrow-derived megakaryocytes, a single cell suspension of bone marrow from wild C57BL/6 mice was collected and cultured in IMDM medium (Gibco, New York, USA) containing 1% penicillin/streptomycin (Gibco, New York, USA) and 20% heat-inactivated fetal bovine serum (Gibco, New York, USA) in the presence of 100 ng/ml recombinant murine thrombopoietin (rmTPO) and 100 ng/ml murine stem cell factor (SCF) for 7 days with or without 10 μM celecoxib. Megakaryocytes were purified and enriched by a 1.5%/3.0% discontinuous bovine serum albumin (BSA) gradient [27 (link)] and prepared for the following experiments.
3
Culturing and Manipulating Mammary Epithelial Cells
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Cells were cultured at 37°C with humidified 5% CO2. Nontransformed hTERT1-HMECs were obtained from the laboratory of Stephen Elledge at Harvard Medical School (Solimini et al., 2012 ) and cultured in Mammary Epithelial Growth Medium (Lonza CC-3150). To reduce background fluorescence in imaging experiments we used the same medium without phenol red (Lonza CC-3153 basal medium supplemented with growth factors and other components from the CC-4136 kit). Medium was refreshed every 24 h during imaging experiments to mitigate phototoxicity and nutrient depletion. Endogenously tagged RB-Clover cells were generated as described in Zatulovskiy et al. (2018) . HEK 293T cells (for producing lentivirus) were cultured in DMEM containing l -glutamine, 4.5 g/l glucose, and sodium pyruvate (Corning), supplemented with 10% fetal bovine serum (FBS; Corning) and 1% penicillin/streptomycin. K562 cells were a gift from Ravi Majeti’s lab at Stanford University and were generally cultured in RPMI 1640 medium (HyClone) supplemented with 10% FBS and 1% penicillin/streptomycin. For 3 wk after single-cell sorting, K562 clones were expanded in IMDM medium (Life Technologies) supplemented with 20% FBS and 1% penicillin/streptomycin.
4
Establishing Ameloblastoma and Cancer Cell Lines
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HAM2 and HAM3 ameloblastoma cell lines were established from a human ameloblastoma tissue, as described previously21 (link). HAM cell lines were maintained at 37 °C in 5% CO2 in keratinocyte-SFM medium (K-SFM; Life Technologies, Carlsbad, CA, USA). DLD1 colorectal cancer cells were purchased from ATCC (Manassas, VA, USA) and cultured at 37 °C in 5% CO2 in DMEM medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5% FBS (AusGeneX, Molendinar, QLD, Australia). MCF10A mammary gland cells were obtained from the Barbara Ann Karmanos Cancer Institute Cell Line Resource (Detroit, MI, USA) and cultured at 37 °C in 5% CO2 in IMDM medium (Life Technologies) supplemented with 10% FBS (Life Technologies). Plasmid DNAs and siRNAs were transfected using Lipofectamine 3000 and Lipofectamine RNAiMAX, respectively (Life Technologies). The transfection of HAM cell lines was performed in IMDM medium supplemented with 10% FBS because K-SFM medium was not suitable for the transfection reaction. D4476, a CK-1 inhibitor, was used at a concentration of 100 μM (Abcam, Cambridge, UK).
5
Thapsigargin-Induced Cytokine Release Assay
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Heparinized human blood was diluted with an equal volume of IMDM medium (Life Technologies) supplemented with 2% normal human A/B serum and plated at 200μl/well in 96-well plates for thapsigargin (Alomone) stimulation. CD4 T cells were plated at 5 x 105 cells/well in 96-well plates with 200μl medium/well. Blood or T cell samples were pre-treated with blockers or inhibitors for 30 minutes prior to stimulation with thapsigargin at 10μM or other concentrations as indicated for 20 hours. Cytokines in culture supernatants were measured as described in cytokine detection section.
6
Cell Line and Primary Cell Culture Conditions for ALL
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For cell lines and their driver oncogenes, see Supplementary Table S1 . BV173, BV173R, 697, and SD1 cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Paisley, UK). DOHH2, NALM6, REH, and SEM cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. SUP-B15 and SUP-B15R cells were cultured in McCoy's 5A medium (Life Technologies) supplemented with 20% FBS and 1% penicillin/streptomycin. RS4;11 cells cultured in α-MEM medium (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin.
Primary CD34+ ALL and normal cells were cultured in IMDM medium (Life Technologies) supplemented with 25% BIT (bovine serum albumin/insulin/transferrin; StemCell Technologies, Vancouver, BC, Canada), L-glutamine, penicillin/streptomycin, and 125 μM 2-mercaptoethanol (Life Technologies). ALL CD34+ cells were cultured in the presence of FLT3-ligand (100 ng/ml), IL-7 (100 ng/ml), and SCF (100 ng/ml), as described.25 (link) Normal CD34+ cells were cultured in a five growth factor cocktail containing FLT3-ligand (100 ng/ml), G-CSF (20 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), and SCF (100 ng/ml) (all growth factors are from PeproTech, Rocky Hill, NJ, USA).
Primary CD34+ ALL and normal cells were cultured in IMDM medium (Life Technologies) supplemented with 25% BIT (bovine serum albumin/insulin/transferrin; StemCell Technologies, Vancouver, BC, Canada), L-glutamine, penicillin/streptomycin, and 125 μM 2-mercaptoethanol (Life Technologies). ALL CD34+ cells were cultured in the presence of FLT3-ligand (100 ng/ml), IL-7 (100 ng/ml), and SCF (100 ng/ml), as described.25 (link) Normal CD34+ cells were cultured in a five growth factor cocktail containing FLT3-ligand (100 ng/ml), G-CSF (20 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), and SCF (100 ng/ml) (all growth factors are from PeproTech, Rocky Hill, NJ, USA).
7
Cell Line Maintenance Conditions
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All the MCL (Jeko-1, Mino and Rec-1) and ABC–DLBCL cell lines (U2932 and RI-1) were maintained in the RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% foetal calf serum (FCS) (Biochrom AG, Berlin, Germany) and 2 mM glutamine (Life Technologies, Gent, Belgium). The GCB–DLBCL cell lines (SU-DHL-6, OCI-Ly1 and OCI-Ly7) were maintained in the IMDM medium (Life Technologies) supplemented with 10% FCS and 2 mM glutamine. Cells were cultured at 37 °C in a humidified 5% CO2atmosphere. All cell lines were obtained from ATCC and regularly tested for mycoplasma contamination. They were authenticated by STR profiling.
8
Directed Differentiation of Skeletal Muscle
9
Maintenance of DLBCL and MCL Cell Lines
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The human GCB-DLBCL cell lines SU-DHL-6, OCI-Ly1, and OCI-Ly7 were maintained in IMDM medium (Life Technologies) supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany) and 2 mM glutamine (Life Technologies, Gent, Belgium). ABC-DLBCL (U2932 and RI-1) and MCL cell lines (Jeko-1, Mino and Rec-1) were maintained in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% FCS and 2 mM glutamine. The murine DLBCL cell line (A20) was maintained in supplemented RPMI-1640 medium with 0.05 mM β-mercapto-ethanol (Sigma-Aldrich). Cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. All cell lines were obtained from ATCC and regularly tested for mycoplasma contamination and checked for authenticity by STR profiling.
10
Maintenance of ALMC-2 and KAS-6/1 cell lines
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The HMCLs ALMC-2 and KAS-6/1 were established in our laboratory and described elsewhere.14 (link), 15 (link) The cells are routinely maintained in IMDM medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal calf serum and 1 ng/ml recombinant IL-6.
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