Four healthy donors of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were purchased from Rooster Bio (MSC-003 lots: 00182, 310,271, 310,267 and 310,263) and used in the experiments with population doubling levels (PDL) between 13.2 and 15. The donors were derived from two females and two males, with ages ranging from 19 to 26 years. Cells were expanded in DMEM high glucose (D6429, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (F4135, Sigma) and 1% penicillin–streptomycin (P4333, Sigma) and maintained at 37℃ and 5% CO2 in a humidified incubator. Peripheral blood mononuclear cells (PBMCs) were purchased from Cell Applications, Inc. (690 PB-100a) and maintained in RPMI-1640 medium (R8758, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (F4135, Sigma), 1% penicillin–streptomycin (P4333, Sigma-Aldrich) and 1% nonessential amino acids—NEAA (M7145, Sigma).
F4135
Manufactured by Merck Group
Sourced in United States, United Kingdom
The F4135 is a laboratory equipment product from Merck Group. It is designed for precise and efficient sample preparation. The core function of the F4135 is to provide controlled temperature and mixing for various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Universal mycoplasma detection kit, by American Type Culture Collection (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Anti cd11b coated microbeads, by Miltenyi Biotec (2 mentions)
Percoll, by GE Healthcare (2 mentions)
Quadromacs separator, by Miltenyi Biotec (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Cytiva (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Skbr3, by American Type Culture Collection (2 mentions)
A2780, by Merck Group (2 mentions)
A2780cis, by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Htb 96, by Corning (1 mentions)
37 protocols using f4135
1
Expansion of Human Bone Marrow Mesenchymal Stem Cells
2
Characterization of Human Cell Lines
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Human osteosarcoma U2OS cells, purchased from the American Type Culture Collection (ATCC; HTB96), were grown in DMEM (Corning; 10-013-CV) supplemented with 10% newborn calf serum (Hyclone; SH30118.03). Human cervical cancer HeLa cells were procured from ATCC (CCL-2) and grown in DMEM (Corning; 10-013-CV) supplemented with 10% fetal bovine serum (F4135; Sigma). INF2 KO U2OS cells were generated in our laboratory using CRISPR/Cas9 and were reported previously (Chakrabarti et al., 2018 (link)). The GFP-Drp1 knock-in U2OS cell line made by CRISPR/Cas9 was described elsewhere (Ji et al., 2017 (link)). The cell line expresses GFP-Drp1 at 50% of the total Drp1 level, with the remaining Drp1 being unmodified (overall Drp1 level unchanged from control cells). All cell lines were cultivated at 37°C with 5% CO2.
3
Isolation and Culture of Neonatal Murine Microglia
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Microglia were isolated from neonatal (P5-P7) wild type mice for plating in Axion MEA plates, culture, and protein as previously described25 (link),71 . Mice were rapidly killed, brains were extracted, and meninges were removed prior to extracting forebrain. Tissue was mechanically dissociated in glass homogenizers and enrichment of live cells was achieved by centrifugation over 20% Percoll (17-0891-02, GE Healthcare). The microglia were selected by anti-CD11b-coated microbeads (130-093-636, Miltenyi) with the QuadroMACs separator following the manufacturer’s recommendations. Cells were manually counted with a hemocytometer using trypan exclusion staining. For co-culture with neurons in Axion MEA plates, 100,000 microglia were added per well. For HPLC, microglia were plated at 56,000/cm2 (link) in a 12-well plate with DMEM supplemented with 10% FBS (F4135, Sigma) and 1% penicillin–streptomycin (15140, Gibco). Microglia purity was determined by FACS and immunostaining, cultured cells were also used for immunoblotting.
4
Culturing and Characterizing Human and Mouse Cell Lines
5
BPH-1 Cell Culture Protocol
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BPH-1 cells were purchased from DMSZ (Cat# ACC-143, RRID: CVCL_1091) in 2005 and were cultured in RPMI-1640 (R8758, Sigma) containing 10% FBS (F4135, Sigma) and 1% antibiotic antimycotic solution (A5955, Sigma). Cells were regularly negative tested for mycoplasma contamination using MycoStrip™ - Mycoplasma Detection Kit (MycoStrip™ 100, InvivoGen).
6
Amino Acid Starvation in Drosophila S2 Cells
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Drosophila S2 cells (R69007, Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Schneider’s medium (SCH, S0146; Sigma, St. Louis, MO, USA) supplemented with 10% insect-tested fetal bovine serum (F4135; Sigma, St. Louis, MO, USA) at 26 °C. S2 cells (between passages 5 and 18) were pelleted at 200 g in a microfuge for 3 min, washed once in fresh Schneider’s medium, and diluted to 106 mL. 1 mL of cell suspension was plated per well in a 12-well plate containing coverslips. Cells were allowed to attach for 1.5 h before starting the treatment. Amino acid starvation was performed in Krebs Ringers bicarbonate buffer (KRB) comprising 0.7 mM NaH2PO4, 15 mM NaHCO3 (sodium bicarbonate, BIC), 120.7 mM NaCl, 4.53 mM KCl, 0.5 mM MgCl and 10 mM glucose at pH 7.4 as described previously [12 (link),13 (link)].
7
Nanoparticle Size in Serum Conditions
8
Characterization of Murine Melanoma Cell Lines
9
Cell Line Validation and Maintenance
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All parental cell lines were from validated sources and procured through the Broad Institute’s Dependency Map Project Cancer Cell Line Encyclopedia banks. SpCas9-expressing cell lines were obtained from the Broad Institute’s Genetic Perturbation Platform. All cell lines were originally obtained from authorized cell line banks including the American Type Culture Collection (ATCC), European Collection of Authenticated Cell Cultures (ECACC), Korean Cell Line Bank (KCLB), Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and the Japanese Riken cell line bank. Cell lines were cultured in RPMI-1640 with L-glutamine and phenol red (Corning 10–040-CV) and 10% heat-inactivated fetal bovine serum (Sigma-Aldrich F4135) in addition to other supplements when indicated at 37°C and 5% CO2. Cell lines were initially thawed and expanded in their native, manufacturer recommended culture media supplemented with Penicillin-Streptomycin (Thermo-Fisher, 15140122). However, if the native media was not RPMI, cells were adapted and maintained in RPMI with 10% fetal bovine serum for all experiments after initial expansion. Cell lines were validated by STR profiling and tested for mycoplasma using the PCR-based Universal Mycoplasma Detection Kit (ATCC 30–1012K).
10
Culturing Human Leukemia and Liver Cancer Cells
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The human chronic myeloid leukemia (CML) cell line K562, as well as a CRISPRi derivative of this cell line constitutively expressing a catalytically inactive Cas9 fused to a KRAB effector domain (dCas9-KRAB)[17 (link)] was kindly provided by the Innovative Genomics Institute, UCSF. K562 cell lines and human hepatocellular carcinoma Huh7 cells (ATCC) were cultured in RPMI 1640 medium (Life Technologies, CARLSBAD, CA, USA) supplemented with 0.2 mM L-glutamine (Glutamax, Life Technologies), 10% FBS (F4135, Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (Penicillin/Streptomycin, 0.1 mg/mL, Gibco) unless otherwise stated. HEK293T cells (UC Berkeley Cell culture facility) were maintained in DMEM (Gibco) supplemented with 10% FBS (Seradigm) and antibiotics unless otherwise stated. Cells tested negative for mycoplasma infection before use.
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