LLC and Py8119 cells were from ATCC. LLC cells were maintained in DMEM (Gibco) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS; Capricorn Scientific), 300 μg/ml L-glutamine (Sigma), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco). For Py8119 cells, the medium was F12K (Gibco) and MITO+ serum extender (1:1000, Corning) was added in addition to the supplements above. eGFP-expressing cell lines (produced as described below) were cultured like the parental cell lines. For LLC/LLC-eGFP tumor implantation, 3×106 cells were injected subcutaneously into the right flank of mice in 200 μl of HBSS. For Py8119/Py8119-eGFP tumors, 106 cells were injected into the left 4th mammary fat pad in 50 μl of HBSS mixed with Growth Factor Reduced Matrigel (Corning) in a 1:1 ratio. Tumor volumes were determined by caliper measurements and calculated using the formula: V = π × (d2 × D)/6, where d is the shortest diameter and D is the longest diameter. Tumor, blood, spleen, bone marrow and lung were collected when the tumor reached maximum 1500 mm3 volume.
Mito serum extender
Manufactured by Corning
Sourced in United States, Germany
The MITO+ Serum Extender is a lab equipment product designed to extend the lifespan of cell culture media. It helps maintain the optimal pH and nutrient levels in the media, supporting the growth and maintenance of cells in in vitro experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Poly d lysine, by Merck Group (3 mentions)
Thermomixer, by Eppendorf (3 mentions)
Collagen 1, by Corning (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
C57bl 6n mice, by Japan SLC (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Bovine pituitary extract, by Corning (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Entero stim intestinal epithelium differentiation medium, by Corning (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Ham s f 12k kaighn s medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
25 protocols using mito serum extender
1
Murine Cancer Models for Tumor Profiling
2
Investigating Antitumor Immunity in Mice
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Six-week-old female C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan) and kept in a specific pathogen-free environment. The animal use proposal and experimental protocols were reviewed and approved by The University of Tokyo Animal Care and Use Committee (ID:P15-125) and all animal procedures were conducted in accordance with institutional guidelines. The YTN2 and YTN16 cell lines [6 (link)] were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL streptomycin, 100 U/mL penicillin (Nacalai Tesque), and MITO+ serum extender (Corning, Corning, NY, USA). Anti-PD-1 (RMP1-14), PD-L1 (10F9G2), CTLA-4 (9H10), and CD8α (53–6.7) mAbs were from Bio X Cell (Lebanon, NH, USA). DimerXI:Recombinant Soluble Dimeric Mouse H-2Db:Ig (H-2Db dimer), DimerXI:Recombinant Soluble Dimeric Mouse H-2Kb:Ig (H-2Kb dimer) were from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-CD3ε, PerCP/Cyanine5.5-conjugated anti-CD4, APC/Cyanine7-conjugated anti-CD8, APC-conjugated anti-IFN-γ and Pacific Blue-conjugated anti-CD45 mAbs were from BioLegend (San Diego, CA, USA).
3
Culturing Murine TNBC Cell Lines
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Firefly luciferase-expressing 4T1 mammary tumor cells that resemble metastatic human TNBC were provided as a kind gift from Prof. Clare Isacke (Breakthrough Breast Cancer Research Center, London, UK) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (both from Sigma-Aldrich, Overijse, Belgium) at 37°C and 5% CO2. Py230 mammary tumor cells, derived from the American Type Culture Collection (ATCC) and reported to model TNBC,18 (link),20 (link) were cultured in Ham’s F-12 K Medium (Thermo Fisher Scientific) supplemented with 5% heat-inactivated FBS (Thermo Fisher Scientific), 0.1% MITO+ Serum Extender (Corning, New York, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich) at 37°C and 5% CO2. Checks for mycoplasma and other bacterial contamination were routinely performed using a PlasmoTestTM mycoplasma detection kit (Invivogen, San Diego, USA). Harvesting of the cells involved washing with phosphate buffered saline (PBS) and incubation with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) for 5 minutes (min). The cells were subsequently centrifuged at 805 × g for 5 min, resuspended in PBS and counted with a Bürker chamber.
4
Induced Astrocyte Differentiation Protocol
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We differentiated HiAs as previously reported (Perriot et al., 2018 (link)). Lt-NES cells were dissociated with trypsin-EDTA, plated on Matrigel (Corning, Corning, NY)-coated T25-flask at 1 × 106 cells in Astrocyte Induction Medium (DMEM/F12; Sigma-Aldrich, St Louis, USA, GlutaMax; Thermo Fisher Scientific, Waltham, MA, 1/100, N2 supplement; Thermo Fisher Scientific, 1/100, 10 ng/ml LIF; Sigma-Aldrich, 10 ng/ml EGF; PeproTech, B27 minus vitamin A; Thermo Fischer Scientific, 1/50) and medium was changed every 3–4 days for 2 weeks. After 2 weeks, cells were trypsinized, and a half volume of cells was plated on Matrigel-coated T25-flasks in Astrocyte Maturation Medium (DMEM/F12, GlutaMax 1/100, B27 minus vitamin A 1/50, 20 ng/ml CNTF; PeproTech) and incubated for 4 weeks, changing the medium every 3 to 4 days. Four weeks later, the cells were removed by trypsin and plated in Matrigel-coated T75 flasks in 10% FBS/DMEM (DMEM, FBS 1/10, GlutaMax 1/100, MITO+ Serum Extender; Corning, 1/1000) and incubated for a week. Finally, the cells were reacted with mitomycin C (Nacalai Tesque, Kyoto, Japan) in 10 μg/ml for 2 hours to inhibit cell proliferation. For cryopreservation, cells were trypsinized, and around 1–2 × 106 cells were spun down and resuspended in CELLBANKER 2 before freezing at −80°C.
5
Establishment and Culture of MamBo Cell Lines
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MamBo cell lines were established from mammary tumors of FVBhuHER2 virgin female mice. Tumors were minced and set in culture with an appropriate medium as previously described [22 (link)]. Cell lines were stabilized and cultured in DMEM (Thermo Fisher Scientific, Monza, Italy) that was supplemented with 20% fetal bovine serum (FBS, Thermo Fisher Scientific), 30 µg/ml bovine pituitary extract (Corning Life Sciences, Glendale, AZ, USA) and 0.5% v/v MITO Serum Extender (Corning). Cell cultures were maintained at 37 °C in a humified 5% CO2 atmosphere.
6
Murine Gastric Cancer Cell Lines
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Six-week-old female C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan). All mice were kept in a specific pathogen-free environment. YTN2 and YTN16 are cell lines established from chemically induced gastric cancers and are maintained in Dulbecco's modified Eagle's medium (DMEM, Nacalai Tesque, Kyoto, Japan) with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, Missouri, USA), 100 µg/mL streptomycin, 100 U/mL penicillin (Wako Pure Chemical) and MITO+ serum extender (Corning, Corning, New York, USA). Antibodies specific for CD4 (GK1.5), CD8α (53–6.7), NK1.1 (PK136), PD-1 (RMP1-14), IL-17A (17F3) and CD16/32 (2.4G2) were all purchased from BioXcell (Lebanon, New Hampshire, USA). DimerX I:Recombinant Soluble Dimeric Mouse H-2Db:Ig, DimerX I:Recombinant Soluble Dimeric Mouse H-2Kb:Ig, APC-conjugated anti-CD103 and PE-CF594-conjugated anti-Ly6G mAbs were from BD Biosciences (Franklin Lakes, New Jersey, USA). FITC-conjugated anti-I-A/I-E and anti-CD3 mAbs, PE-conjugated anti-mouse IgG1, anti-IL-17, anti-NK1.1 and anti-CD64 mAbs, PerCP/Cy5.5-conjugated anti-CD11b, anti-CD4, anti-LAG-3 and anti-TCRγ/δ, APC-conjugated anti-IFNγ, anti-TIM-3 and anti-CTLA-4, PE/Cy7-conjugated anti-Ly6C and anti-PD-1, APC/Cy7-conjugated anti-CD8α and anti-CD45 and Pacific Blue-conjugated anti-CD45 mAbs were all from BioLegend (San Diego, California, USA).
7
Intestinal Epithelium Differentiation Protocol
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MEM with Earle's salts, DMEM, fetal bovine serum (FBS), trypsin‐EDTA, and antibiotics were purchased from Biochrom GmbH (Berlin, Germany). Entero‐STIM Intestinal Epithelium Differentiation Medium and MITO+ Serum Extender were purchased from Corning (Wiesbaden, Germany). Hesperetin, peltatoside, hyperoside, kaempferol, ellagic acid, isoquercitrin, quercitrin, phloretin, (−)‐epicatechin, epigallocatechin, epicatechin gallate, procyanidin B1, procyanidin B2, epigallocatechin gallate, gallocatechin, and morine were obtained from Extrasynthese (Genay Cedex, France). Phloridzin, chlorogenic acid, caffeic acid, quercetin, (+)‐catechin, gallic acid, 2‐mercaptoethanol, NaCl, MgSO4, KCl, CaCl2, HEPES, and xylitol were purchased from Sigma‐Aldrich (Schnelldorf, Germany). Guaijaverin and avicularin were obtained from Glentham Life Sciences (Corsham, United Kingdom). Purified and concentrated GLE (cGLE) was provided by Belan GmbH (Wels, Austria).30 Guava fruit puree for preparation of fruit extracts was obtained from Tradework BV (Rotterdam, The Netherlands), commercially available GLE and apple extract (AE) were purchased from Pfannenschmidt (Hamburg, Germany). Transwell inserts (12 mm, collagen‐treated, 0.4 μm pore size) and 12‐well plates were obtained from Corning (Wiesbaden, Germany).
8
Culturing Mammary Carcinoma Cell Lines
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Cell line MAMBO89, established from a mammary carcinoma of huHER2 transgenic mouse,38 (link) was cultured in DMEM (Thermo Fisher Scientific) additioned with 20% fetal bovine serum (FBS, Thermo Fisher Scientific), 30 µg/ml bovine pituitary extract (Corning) and 5 µl/ml MITO+ SERUM EXTENDER (Corning). MAMBO89 cell line was monitored for murine origin by PCR and for full-length HER2 transgene expression by PCR and flow cytometry. Human ovarian cancer cell line SK-OV-3 and human breast cancer cell lines MDA-MB-453, BT-474 and SKBR3 (all from American Type Culture Collection, ATCC) were kindly given by Dr. Serenella M. Pupa (Istituto Nazionale dei Tumori, Milan, Italy). SK-OV-3, MDA-MB-453 and BT-474, were authenticated by DNA fingerprinting on the 11th November 2010 (performed by DSMZ, Braunschweig, Germany). HCC1954, MDA-MB-231 and BT-474 C5 cell lines were purchased from ATCC. Cells were routinely cultured in RPMI medium (Thermo Fisher Scientific) supplemented with 10% FBS. Human rhabdomyosarcoma cell line SJ-RH4, lacking HER2 expression, was cultured in DMEM additioned with 10% FBS. Cell lines were maintained at 37°C in a humidified 5% CO2 atmosphere, except SJ-RH4, which was kept at 7% CO2.
9
Melanoma Cell Culture Under Hypoxia
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Human cutaneous melanoma cell line C-8161 [32 (link)] was cultured in RPMI 1640 medium containing 2-mM glutamine, 50 U/mL of penicillin (Gibco, Minneapolis, MN, USA), 100 mg/mL of streptomycin (BD Biosciences, Bedford, MA, USA), and 10% fetal bovine serum (Gibco, Minneapolis, MN, USA), supplemented with 1× MITO + serum extender (Corning Inc., Lowell, MA, USA). Cells were cultured with plasma from randomized patients with OSA at a concentration of 10% or with rPSPC1 (2500 pg/mL), and/or rTGFβ (50 pg/mL), and/or αPSPC1 (1 μg/mL) either under normoxia or IH conditions. IH and normoxia conditions were the same as those used for the IH model.
10
Cell Culture Conditions for Esophageal and Gastric Cancer Lines
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Human esophageal cancer cells lines OE19, ESO26, OACP4C, and SKGT4 cells were grown in RPMI-1640 medium (Corning, #45000-396). Flo-1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Corning, #45000-304). Both media were supplemented with 10% FBS (Omega Scientific, #FB-02) and 1% penicillin-streptomycin sulfate (Gibco, #10378016). Murine gastric cancer cells, YTN 5 and YTN16 were kindly provided by Dr. Sachiyo Nomura at The University of Tokyo. YTN5 and YTN16 cells were cultured in DMEM (Corning, #45000-304), with 10% heat-inactivated fetal bovine serum (Omega Scientific, #FB-02) and 1% penicillin-streptomycin sulfate (Gibco, #10378016), 1X GlutaMAX (Gibco, #35050-061) and MITO+ serum extender (Corning, #355006). All cultures were maintained in a 37 °C incubator supplemented with 5% CO2. All the cell lines were tested for mycoplasma and verified by us using short tandem repeat analysis.
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