Human ALL cell lines (DND41, HPB-ALL, MOLT-4, RPMI-8402, CCRF-CEM, JURKAT) and AML cell lines (KG-1, HL-60, NOMO-1, KASUMI-1, MOLM-13, THP-1, FKH-1) were grown in RPMI1640 (Lonza) supplemented with 10% Fetal Bovine Serum (Invitrogen), 2 mM L-glutammine,100 U/mL penicillin and 100 μg/mL streptomycin (GIBCO), SEM cell line was grown in Iscove medium (Sigma) supplemented with 20% FBS (Invitrogen). OCI-AML3 and OCI-AML6 were grown in Alpha-MEM (Lonza). UCSD-AML1 was supplemented with GM-CSF 10 ng/ml (Sigma) and MM-6 (Mono-Mac-6) was supplemented with OPI (Sigma). Primary cells derived from AML patients were cultured with Chang Marrow medium (Technogenetics). All cell lines were grown at 37°C in a humidified atmosphere of 5% CO2. L-Asp (Erwinase) was purchased by Jazz Pharmaceutical. The patients analyzed were enrolled in the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) 2002/01 AML Study [2 (link)] after obtaining written informed consent from the parents according to the Declaration of Helsinki. Pt1 was classified as AML FAB M4 (Acute myelomonocytic leukemia) with chromosome 7 disomy; Pt 2 was a AML FAB M2 (Acute myeloblastic leukemia with maturation) with disomy of chromosome 7; Pt 3 was a AML FAB M2 with monosomy of chromosome 7.
Iscove medium
Manufactured by Merck Group
Sourced in Germany
Iscove medium is a cell culture medium formulation developed by Iscove and Melchers. It is commonly used for the cultivation and maintenance of various cell types, including hematopoietic cells and other cell lines. The medium provides a balanced solution of nutrients, vitamins, and other essential components required for cell growth and proliferation.
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Lab products found in correlation
Penicillin streptomycin, by Harvard Bioscience (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Lonza (1 mentions)
Gm csf, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
X vivo 10, by Lonza (1 mentions)
Automacs separator, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Dnase 1, by Roche (1 mentions)
Glutamine, by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Liberase, by Roche (1 mentions)
Y 27632, by Merck Group (1 mentions)
Anti cd45 microbeads, by Miltenyi Biotec (1 mentions)
Akta pure, by Cytiva (1 mentions)
Ionomycin, by BD (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
6 protocols using iscove medium
1
Acute Leukemia Cell Culture Protocols
2
Isolation and Culture of Thymic Epithelial Cells
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Thymus was isolated from 4‐ to 6‐week‐old WT mice. Thymus was cleaned up from fat and stromal tissues under the microscope and then digested at 37°C (three steps of 5 minutes) with a solution containing Liberase (Roche, Germany) and DNAse I (Roche). Digested thymus was recovered in Iscove medium (Sigma–Aldrich, Germany) supplemented with 10% fetal bovine serum (FBS; Euroclone, Italy), 1% penicillin/streptomycin (Lonza, Switzerland), and 2% glutamine (Lonza). TECs were further enriched by depleting CD45+ cells with the AutoMACS Separator (Miltenyi Biotec, Germany) after incubating thymic cells with anti‐CD45 microbeads (Miltenyi Biotec). The purity of enriched TECs (CD45‐EpCam+) was then checked by multicolor flow cytometric analyses. Enriched TECs were cultured in CnT‐57.S (CELLnTEC Advanced Cell Systems, Switzerland) supplemented with Rho‐associated, coiled‐coil containing protein kinase (ROCK) inhibitor 1 μg/ml (Y‐27632, Merck Millipore) for 7–9 days after isolation; afterward, culture medium was switched to X‐VIVO10 (Lonza) supplemented with the ROCK inhibitor.
3
Purification of Anti-Mouse IgG2a Antibodies
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HB-79 (producing anti H-2Kd/H-2Dd mouse IgG2a) and HB-27 (producing anti H-2Ld mouse IgG2a) hybridoma cell lines were purchased from ATCC and grown in Iscove medium (Sigma) supplemented with 10% FBS. Hybridoma were then adapted to protein-free PFHM medium (Thermo) for expansion and conditioning. Once cells were dead, the medium containing immunoglobulins was centrifuged and filtered to be run on a MabSelect Sure (ProteinA) column (Cytiva) mounted on Akta Pure (Cytiva). IgGs were then eluted at acid pH and dialyzed against physiologic storage buffer.
4
Splenocyte Effect on Fibroblast Collagen
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To determine the impact of splenocytes on collagen production in fibroblasts, splenocytes of the different experimental groups were co-cultured with murine C4 fibroblasts. According to Van Linthout et al. (11 (link)), fibroblasts were plated at a density of 10,000 cells per well in Iscove Basal Medium (Sigma-Aldrich) containing 10% FBS and 1% penicillin/streptomycin (both Biochrom). Twenty-four hours later, isolated splenocytes were added at a ratio of 1 to 10 (fibroblasts to splenocytes) in Iscove medium (Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin (both Biochrom) in the presence of 50 ng/ml of PMA and 500 ng/ml of Ionomycin (both BD Biosciences). After 24-h co-culture, splenocytes were removed, and fibroblasts were fixed with cold methanol. Subsequently, Sirius red staining was performed. For photometric analyses at 540 nm, the Spectra Max 340PC microplate reader (Molecular Device GmbH) was used.
5
Murine Fibroblast Stimulation Assay
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Murine C4 fibroblasts, a murine fibroblast cell line derived from embryonic BALB/c mice by SV40 infection in vitro36 (link), were cultured in Iscove medium (Sigma Aldrich, Munich, Germany) supplemented with 10% FCS (Biochrom) and 1% penicillin/streptomycin (Biochrom). For stimulation experiments, 60,000 cells/well were plated in 12-well-plates. One day after plating, cells were washed once with phosphate buffered saline (PBS; Thermo Fisher Scientific, Waltham, Massachusetts, USA), and starvation medium (Iscove medium + 0.05% FBS + 1% P/S) was added. After 24 hours (h), cells were stimulated with or without 5 ng/mL TGF-β1 (Peprotech, Hamburg, Germany), 10 ng/mL IFN-γ (Peprotech) or both in starvation medium. 72 h after stimulation, the supernatant was harvested, centrifuged (10 minutes (min) at 4 °C, 12000 × g) and aliquoted to further investigate chemokine secretion and perform chemotaxis assays.
6
Agnosphere Isolation and Propagation Protocol
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Agnospheres were isolated and cultured as described previously (Verginelli et al, 2021 (link)). Briefly, tumor samples were digested with collagenase I (Gibco), and after filtration, single‐cell suspensions were resuspended in culture medium [Dulbecco's Modified Eagle's Medium: F12 (DMEM; Sigma) supplemented with N2 supplement (Life Technologies‐GIBCO), BSA 0.5% (Sigma), Heparin 4 μg/ml (Sigma), 2 mM Glutamine (Sigma), and Penicillin–Streptomycin (EuroClone)]. Ultralow‐attachment flasks (Corning, cat. CLS3814) were used in case of AS901. The same medium composition was used for further propagation of the agnospheres; when subculturing, agnospheres dissociation was mechanically achieved by pipetting and by trypsin treatment. MCF7 and HEK‐293T cells were provided by American Type Culture Collection (ATCC) and cultured in DMEM or Iscove medium (Sigma‐Aldrich), respectively, supplemented with 10% fetal bovine serum (FBS; Euroclone), 2 mM Glutamine (Sigma‐Aldrich), and Penicillin–Streptomycin (EuroClone). The cells were periodically checked for mycoplasma contamination.
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