Acetyl-11-keto-β-boswellic acid—AKBA (Santa Cruz, CA, USA) is characterized by the following properties (according to manufacturer): molecular formula C32H48O5, MW 512.72, purity (HPLC) 99.6% and purity (TLC) 99.6%. AKBA was prepared in stocks of 200 mM in dimethyl sulfoxide (DMSO), stored at -20°C and was thawed and diluted in a cell culture medium immediately before treatment. Antibiotics (penicillin, streptomycin, amphotericin) and a kit for colorimetric tetrazolium salt assay (XTT) for cell survival were obtained from Biological Industries (Beit-Haemek, Israel). Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Life Technologies (Rehovot, Israel). Antibodies against PARP, NF-ĸB, p65, IĸB-α, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein isothiocyanate-conjugated annexin V (Annexin V-FITC) with propidium iodide (PI) was received as a Tali apoptosis kit (Life Technologies, Rehovot, Israel).
Amphotericin
Manufactured by Sartorius
Sourced in Israel
Amphotericin is a antifungal agent used in laboratory settings. It is a broad-spectrum polyene macrolide antibiotic that functions by binding to ergosterol, a component of fungal cell membranes, leading to increased cell permeability and cell death.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Sartorius (15 mentions)
Penicillin, by Sartorius (11 mentions)
L glutamine, by Sartorius (8 mentions)
Fetal bovine serum (fbs), by Sartorius (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Sodium pyruvate, by Sartorius (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Gentamycin sulfate, by Sartorius (2 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (2 mentions)
Tali apoptosis kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Arpe 19, by RIKEN BioResource Center (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Hct116 ccl 247, by American Type Culture Collection (1 mentions)
Rhegf, by R&D Systems (1 mentions)
B 27 serum free supplement, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Poly 2 hydroxyethyl methacrylate, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
19 protocols using amphotericin
1
Characterization of AKBA Compound
2
ARPE-19 Cell Culture Conditions
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Human retinal pigment epithelial cells (ARPE-19) were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). Dulbecco’s modified Eagle’s medium (Gibco Laboratories, Grand Island, NY, USA) with 10% w/v fetal bovine serum (Gibco Laboratories, NY, USA), 100 units/mL penicillin (Biological Industries, Beit Haemek, Israel), 0.1 mg/mL streptomycin (Biological Industries, Beit Haemek, Israel), and 2.5 × 10−4 mg/mL amphotericin (Biological Industries, Beit Haemek, Israel) was used to prepare cell media. The cells were incubated in 10 cm dishes at 37 °C and 5% CO2.
3
HCT116 Colon Carcinoma Cell Culture
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HCT116 (CCL-247) (human colon carcinoma) cell line was purchased from American Type Culture Collection (ATCC) (Rockville, MD, United States). Cells were cultured in Dulbecco’s modified Eagle’s (DMEM) medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 units/mL of penicillin, 100 μg/mL of streptomycin and amphotericin (Biological Industries, Beit HaEmek, Israel) and grown in plastic flasks (Rutherford, NJ, United States).
4
Generating Tumor Spheroids for Microenvironment Studies
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Six-well plates were incubated overnight on a rocker with 1.2% poly(2-hydroxyethyl methacrylate) (Cat# P3932; Sigma) diluted in ethanol. mCherry-expressing MCF-7 cells were plated in the coated wells, in DMEM/F12 medium supplemented with 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin (all from Biological Industries), 0.4% BSA (Cat# 0332-TAM; Amresco, Solon, OH, USA), B-27 serum-free supplement (Cat# 17504044; Gibco, Life technologies, Grand island, NY, USA), 20 ng/ml rh-basic FGF (Cat# 100-18B; PeproTech), 20 ng/ml rh-EGF (Cat# 236-EG; R&D systems, Minneapolis, MN, USA), and 5 µg/ml insulin (Cat# I9278; Sigma). After 72 h, tumor spheroids were collected, centrifuged (1,200 rpm for 7 min, + 4°C) and resuspended in the different MSC-derived CM or the respective control media (as described above). Tumor spheroids were photographed daily using fluorescent microscopy.
5
Cell Culture Conditions for Adenocarcinoma and Kidney Cell Lines
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Human lung adenocarcinoma cell line A549 (ATCC CCL-185) and human embryonic kidney 293 T cells (ATCC CRL-3216) were cultured in DMEM (GIBCO, 12800-017) and Jurkat T cells (ATCC TIB-152) were cultured in RPMI-1640 (Hyclone, SH30027.02). All media contained penicillin, streptomycin, amphotericin (Biological Industries, 03-033-1B) and 10% fetal bovine serum (FBS; Biological Industries, 04-001-1A). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2. 293T and Jurkat T cells were used for lentivirus production and phagocytosis assay, respectively.
6
Human Breast Cancer Cell Cultures
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Human breast adenocarcinoma MDA-MB-231 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM l -glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin (all purchased from Biological Industries, Beit Haemek, Israel). In this study, we used two spontaneous variants of MDA-MB-231 cells, expressing either low or high levels of endogenous CCL2 [22] (link), [27] (link), [40] (link), [41] (link), [53] (link) (the latter cells were kindly provided by Dr Gilles, University of Liège, Liège, Belgium). Human embryonic kidney–293 (HEK 293) cells were maintained in DMEM as above. Chinese hamster ovary (CHO) cells were also used in the study (kindly provided by Prof. Vlodavsky, Technion, Haifa, Israel): CHO-K1 and CHO-pgsA-745 cells were cultured in DMEM and Roswell Park Memorial Institute (RPMI) medium, respectively, supplemented as above [with the addition of 1 mM sodium pyruvate (Biological Industries)]. Before different experimental procedures, the various cell lines were transferred to their corresponding serum-free media, except for HEK 293 cells that were continuously grown in their serum-including medium.
7
Breast Cancer Cell Lines for Research
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Human breast tumor MDA-MB-231 cells were obtained from ATCC (Manassas, VA, USA) and MCF-7 cells were kindly provided by Prof. Kaye, Weizmann Institute of Science, Rehovot, Israel. The MCF-7 cells were authenticated on the basis of published characteristics of MCF-7 cells [76 (link),77 (link)] by verifying that they express an active estrogen receptor alpha, respond to estrogen, express low levels of ErbB2, form tumors upon supplementation of estrogen and matrigel, and have low metastatic potential. These cells were grown in enriched Dulbecco’s modified Eagle’s medium (DMEM), including 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin, and 4 mM l -glutamine (Biological Industries, Beit Ha’emek, Israel).
8
Culturing Mouse and Human Cell Lines
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NIH3T3 (Mouse embryo fibroblast), RAW (RAW267.4, Murine monocytes/macrophages-like) and 293FT (Human embryonal kidney) were obtained from ATCC, Manassas, Virginia, USA (CRL-1658, TIB-71 and CRL-3216, respectively). These cell lines were maintained in DMEM supplemented with 10% FCS, 2.5 μg/ml Amphotericin and 50 μg/ml Gentamycin Sulfate (Biological Industries, Beit-Haemek, Israel). Mouse iPS cell line (miPS-B6-GFP) was provided by Prof. Lior Gepstein. Undifferentiated colonies were cultured on mitotically inactivated mouse embryonic fibroblasts (MEF) feeder layer, as previously described [11 (link)]. Cells were maintained in DMEM supplemented with 15% FCS (Biological Industries), 0.1% leukemia inhibitory factor (LIF) (Millipore), 1mM L-glutamine, 0.1mM Mercaptoethanol, and 1% nonessential amino acid stock (all from Invitrogen).
9
Expansion and Characterization of Human Mesenchymal Stem Cells
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Human BM-derived MSCs were purchased from Lonza (Cat# PT-2501; Lonza, Walkersville, MD, USA). The cells were validated as MSCs by Lonza, by marker criteria (positive for CD44, CD29, CD105, and CD166; negative for CD45, CD14, and CD34) and differentiation to adipogenic, chondrogenic, and osteogenic lineages. MSCs of six different donors were used in the study. The cells were thawed in MSC growth medium (MSCGM; Cat# PT-3001; Lonza) and then were subcultured every 5–7 days, for up to 10 passages, in MSCGM or enriched Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries, Beit Ha’emek, Israel), including 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin, and 4 mM l -glutamine (all from Biological Industries).
10
Cytotoxicity Assay with PectaSol-CMCP
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PectaSol-CMCP (ecoNugenics, Santa Rosa, CA) was prepared in a stock of 25 mg/mL
in H2O, stored at −20°C, thawed and diluted in a cell culture medium
immediately before treatment. Antibiotics (penicillin, streptomycin,
amphotericin) and a kit for XTT-based cell proliferation assay were obtained
from Biological Industries (Beit-HaEmek, Israel). Dulbecco’s modified Eagle’s
medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies
(Rehovot, Israel). Propidium iodide (PI) was obtained from Sigma-Aldrich (St.
Louis, MO). Antibodies for western blot analysis were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
in H2O, stored at −20°C, thawed and diluted in a cell culture medium
immediately before treatment. Antibiotics (penicillin, streptomycin,
amphotericin) and a kit for XTT-based cell proliferation assay were obtained
from Biological Industries (Beit-HaEmek, Israel). Dulbecco’s modified Eagle’s
medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies
(Rehovot, Israel). Propidium iodide (PI) was obtained from Sigma-Aldrich (St.
Louis, MO). Antibodies for western blot analysis were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
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