Clonogenic assay was realized with cells seeded into 35 mm petri dish in semi-solid methylcellulose medium (Methocult™ M3231 for murin cells or Methocult™ H4230 for human cells, Stemcell Technologies). Cells were treated with the drugs for 72 h and then centrifugated. The pellet was resuspended in Iscove's modified Dulbecco medium (Lonza) supplemented with 2% fetal calf serum and 50 U/ml penicillin, 50 mg/ml streptomycin at 10,000 cells/ml, and cell suspension was added to methylcellulose medium (1000 cells/1ml/dish) and left at 37 °C and 5% CO2. Colony forming efficiency was determined after 7 days using Leica DMI8 inverted microscope (Leica Microsystems) and quantified using Image J software.
Iscove s modified dulbecco medium
Manufactured by Lonza
Sourced in Switzerland
Iscove's modified Dulbecco medium is a cell culture medium used to support the growth and maintenance of various cell types, including hematopoietic and lymphoid cells. It is a modification of the original Dulbecco's Modified Eagle's Medium (DMEM), designed to enhance the culture of specific cell populations.
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Lab products found in correlation
Flt3 l, by ProSpec (2 mentions)
Pcr mycoplasma detection set, by Takara Bio (2 mentions)
Interleukin 3 (il 3), by ProSpec (2 mentions)
Interleukin 7 (il 7), by ProSpec (2 mentions)
Stem cell factor (scf), by ProSpec (2 mentions)
Methocult m3231, by STEMCELL (1 mentions)
Methocult h4230, by STEMCELL (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Hyclone, by Cytiva (1 mentions)
Human il 3, by Thermo Fisher Scientific (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Poloxamer 338, by BASF (1 mentions)
Human tpo, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Gemini Bio (1 mentions)
Streptomycin, by Gemini Bio (1 mentions)
Glutamine, by Gemini Bio (1 mentions)
6 protocols using iscove s modified dulbecco medium
1
Clonogenic Assay for Cell Viability
2
Culturing HEK293T and U87/CD4+CCR5+ Cells
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HEK293T cells (ATCC® CRL-3216™)were cultured in Dulbecco’s Modified Eagle Medium without HEPES (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% (v/v) inactivated fetal calf serum (FCS; HyClone, Cytiva, Marlborough, MA, USA), penicillin (100 U/mL) and streptomycin (100 µg/mL) (Invitrogen, Carlsbad, CA, USA) and maintained in a humidified 10% CO2 incubator at 37 °C. U87/CD4+-CCR5+ cells (NIH AIDS reagent program) [19 (link)] were cultured in Iscove’s modified Dulbecco medium (Lonza, Basel, Switzerland) supplemented with 10% (v/v) inactivated FCS, penicillin (100 U/mL) and streptomycin (100 µg/mL), treated once a month with G418 (Sigma-Aldrich St. Louis, MO, USA) and maintained in a humidified 5% CO2 incubator at 37 °C.
3
Eosinophil Differentiation from Cord Blood CD34+ Cells
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CD34+ cells purified from human umbilical cord blood (obtained from the New York Blood Center, New York, NY, USA) were cultured in Iscove’s modified Dulbecco medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 10U/mL penicillin and 10 μg/mL streptomycin at 0.3 × 106 cells/mL. To induce eosinophil differentiation, the cells were first expanded in SCF (50 ng/mL), Flt3-L (50 ng/mL), TPO (50 ng/mL), and GM-CSF (0.1 nM) for the first 4 days. Thereafter, cells were cultured in only IL-3 and IL-5 (0.1 nM each), with medium being replenished every 3–4 days. The differentiation stage of the culture was determined by assessing the percentage of mature eosinophils using differential counts of cultured cells stained with Fast Green/Neutral Red. All cytokines were purchased from R&D Systems (Minneapolis, MN, USA).
4
Culture and Maintenance of Ph+ Leukemia Cells
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Cell lines were tested for mycoplasma contamination (PCR Mycoplasma detection set, Takara Bio Inc., Japan) every 6 months. BV173 (CML- lymphoid blast crisis cell line) and SUP-B15 (Ph+ ALL cell line) cells were cultured in Iscove Modified Dulbecco’s Medium (Lonza Inc., Basel, Switzerland) supplemented with 10% heat-inactivated FBS, 1% penicillin- streptomycin, 1% L-glutamine, at 37°C, 5% CO2.
Primary Ph+ adult ALL (n=6) or CML-lymphoid blast crisis (n=1) cells derived from the peripheral blood (n=2) or the bone marrow (n=5) (average 77% blasts; range: 52–98%) were kindly provided by Dr Michael Caligiuri (Ohio State University, Columbus, OH) and Dr Martin Carroll (University of Pennsylvania, Philadelphia, PA) and were maintained in SFEM (Stem Cell Technology, Vancouver, Canada) supplemented with IL-3 (10 ng/ml), IL-7 (10 ng/ml), Flt3-L (20 ng/ml) and SCF (30 ng/ml) (ProSpec, Israel).
Primary Ph+ adult ALL (n=6) or CML-lymphoid blast crisis (n=1) cells derived from the peripheral blood (n=2) or the bone marrow (n=5) (average 77% blasts; range: 52–98%) were kindly provided by Dr Michael Caligiuri (Ohio State University, Columbus, OH) and Dr Martin Carroll (University of Pennsylvania, Philadelphia, PA) and were maintained in SFEM (Stem Cell Technology, Vancouver, Canada) supplemented with IL-3 (10 ng/ml), IL-7 (10 ng/ml), Flt3-L (20 ng/ml) and SCF (30 ng/ml) (ProSpec, Israel).
5
Transduction of CD34+ Hematopoietic Stem Cells
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PB CD34+ samples were obtained from healthy donors from plerixafor and G-CSF mobilization. Cells were thawed and plated at 1 × 106 cells/mL on non-tissue culture-treated 96-well plates pre-coated with retronectin (20 μg/mL; Takara Shuzo). Cells were pre-stimulated for 24 h in X-Vivo 15 medium (Lonza) supplemented with 1× glutamine, penicillin, and streptomycin (Gemini Bio-Products), human Flt-3 ligand (50 ng/mL), human SCF (50 ng/mL), human TPO (50 ng/mL), and human IL-3 (20 ng/mL) (cytokines: PeproTech). CD34+ cells were transduced with concentrated viral supernatants at transduction concentrations of 2 × 105 TU/mL, 6 × 105 TU/mL, 2 × 106 TU/mL, and 6 × 106 TU/mL with transduction enhancer Poloxamer 338 (1 mg/mL) (BASF, Ludwigshafen, Germany). At 24 h after transduction, cells were washed and plated under myeloid culture conditions. Cells were cultured for 2 weeks after transduction in basal bone marrow media consisting of Iscove-modified Dulbecco’s medium (Lonza) 1× glutamine, penicillin, and streptomycin (Gemini Bio-Products), 20% fetal bovine serum, and 0.52% BSA (Sigma-Aldrich) supplemented with human SCF (25 ng/mL), human IL-3 (5 ng/mL), and human IL-6 (10 ng/mL).
6
Culture and Maintenance of Ph+ Leukemia Cells
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