Human leukemia cell lines Jurkat, HL-60, and K-562 were obtained from ATCC. The normal bone marrow cell line (HS-5) were purchased from the American Type Culture Collection. The bone marrow cell line and leukemia cells were cultured in alpha-minimal essential medium (ThermoFisher). HEK293T cells were cultured in Rosewell Park Memorial Institute 1640 (ThermoFisher). All culture medium was supplied with 10% fetal bovine serum (ThermoFisher), 100 units/ml penicillin and streptomycin (Gibco). The cells were cultured at 37 °C and 5% CO2. To analyze the proliferation rate of the cells, cells were seeded at 1 × 104 or 1 × 103 cells/ml in 10-cm dishes and the cell number was counted every day.
Alpha minimal essential medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alpha-minimal essential medium is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It provides the basic nutritional components required for cellular proliferation and survival.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle s media, by Thermo Fisher Scientific (2 mentions)
Ham s f 12 modified essential eagle s media, by Thermo Fisher Scientific (2 mentions)
Ht1080, by American Type Culture Collection (2 mentions)
Anti foxp3 fitc, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm solution, by BD (2 mentions)
Anti il 17 fitc, by Thermo Fisher Scientific (2 mentions)
Anti foxp3 pe, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Tesr1, by STEMCELL (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Anti human cd73 efluor 450, by Thermo Fisher Scientific (2 mentions)
Anti human cd90 thy 1 apc, by Thermo Fisher Scientific (2 mentions)
Geltrex, by STEMCELL (2 mentions)
Human pdgf ab, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Jurkat, by American Type Culture Collection (1 mentions)
26 protocols using alpha minimal essential medium
1
Leukemia Cell Line Cultivation and Proliferation
2
Isolation and Characterization of Rat BM-MSCs
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Isolation of rat BM-MSCs was performed as described previously9 (link). In brief, after male Sprague Dawley rats (Sankyo Lab Service Corp.) were sacrificed, BM was flushed out from removed tibias and femurs. Marrow was cultured in alpha-minimal essential medium (Thermo Fisher Scientific, Lafayette, CO, USA) with 15% fetal bovine serum (FBS) (CCB, NICHIREI BIOSCIENCE, Tokyo, Japan) and 1% penicillin streptomycin (PS) (Thermo Fisher Scientific). The medium was first changed 24 h after seeding and then twice weekly. BM-MSCs that were harvested after up to three passages and that were positive for CD90 and negative for CD45 and CD11b44 (link) were used for further experiments.
3
Culturing Diverse Cell Lines for Research
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The human fibrosarcoma cell line HT1080 (CCL-121) was purchased from the American Type Culture Collection, and human embryonic kidney cells expressing the large-T antigen (HEK293T) were a kind gift from Dr. Ashis Basu (University of Connecticut). Human cells were cultured in Dulbecco’s modified Eagle’s media (Life Technologies, Grand Island, NY) supplemented with 9% fetal bovine serum (Atlanta Biologics, Atlanta, GA). Chinese hamster AA8 51D1 cells were a kind gift from Professor Claudia Wiese (Colorado State University) in which the Rad51D gene was knocked out using a gene-targeting vector [55 (link)]. Rad51D is a paralog of Rad51 which contributes to the HR pathway in vertebrates [56 (link)]. These cells were cultured in alpha minimal essential medium (Life Technologies, Grand Island, NY) supplemented with 9% fetal bovine serum. Chinese hamster lung fibroblast cell line V79 (GM16136) were obtained from the Coriell Institute for Medical Research (Camden, NJ) and cultured in Ham’s F-12 modified essential Eagle’s media (Life Technologies, Grand Island, NY) supplemented with 9% fetal bovine serum. All cells were maintained in a humidified atmosphere of 5% carbon dioxide, 95 % air, at 37 °C.
4
Culturing Diverse Cell Lines for Research
5
Osteoclast Differentiation Assay
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Cells were treated with different concentrations of AGP (0 to 10 μM). The differentiation of osteoclasts from RAW 264.7 cells was induced by adding 50 ng/mL recombinant murine RANKL in alpha-minimal essential medium (Life Technologies) with 10% FBS for 5 days. After treatments, the cells were fixed with 4% paraformaldehyde and stained for the osteoclast enzyme marker, tartrate-resistant acid phosphatase (TRAP) using an acid phosphatase kit (Life Technologies). In our study, TRAP-positive, multinucleated cells were counted as osteoclasts only if the cells showed more than three nuclei.
6
Osteoclast Generation from Mouse Bone Marrow
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Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Burlingame, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate the floating and adherent cells. Nonadherent cells were washed away, and preosteoclasts were further cultured in the presence of 10 ng/mL macrophage colony-stimulating factor (M-CSF), 100 ng/mL receptor activator of RANKL (PeproTech, London, UK) and NST-1s for 4 days to generate osteoclasts. The medium was changed every 2 d. Osteoclasts were generated after 8–10 days.
7
Osteoclast Generation from Mouse Bone Marrow
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Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate floating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8–10 days.
8
Osteoclast Differentiation from CD14+ Monocytes
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CD14+ monocytes (1×106 cells) from human PBMCs were plated in 24-well (Nunc) and cultured in alpha-minimal essential medium (Invitrogen) containing antibiotics and 10% heat-inactivated foetal bovine serum to separate floating and adherent cells. Nonadherent cells were removed by washing with PBS, and preosteoclasts were cultured in the presence of 25 ng/ml M-CSF and 30 ng/ml RANKL (PeptoTech, London, UK). To investigate the impact of oxLDL-C and IL-22 on osteoclast differentiation, 10 μg/ml oxLDL-C, 10 or 50 ng/ml IL-22, and 10 μg/ml oxLDL-C plus 10 or 50 ng/ml IL-22 were added to culture media. Additionally, to investigate the impact of IL-22 blocking agent on oxLDL-C and IL-22 induced osteoclastogenesis, 10 μg/ml oxLDL-C + 50 ng/ml IL-22, and 10 μg/ml oxLDL-C + 50 ng/ml IL-22 + 2 μg/ml IL-22 blocking Ab (16-7222-82; eBiosciences) added condition of in vitro osteoclast differentiation experiments were performed. The medium was changed every 3 days. Osteoclasts were generated after 14 days. A commercial TRAP kit (Sigma-Aldrich, St. Louis, MO, USA) was used according to the manufacturer's instructions; however, counterstaining with haematoxylin was not performed. TRAP-positive multinuclear cells containing 3 or more nuclei were counted as osteoclasts. Three independent person counted multinucleated TRAP-positive cells, and experiment conditions were blinded.
9
Quantifying Th17 and Treg Cells by Flow Cytometry
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Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
10
Quantifying Th17 and Treg Cells by Flow Cytometry
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Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
In vitro osteoclastogenesis and tartrate-resistant acid phosphatase staining Bone marrow cells from mouse femurs were cultured in alpha-minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics and 10% heat-inactivated fetal bovine serum to separate oating and adherent cells. Nonadherent cells were removed by washing with media, and preosteoclasts were cultured in the presence of 10 ng/mL macrophage colony-stimulating factor, 100 ng/mL receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) (PeptoTech, London, UK), and L. sakei for 4 days to generate osteoclasts. The medium was changed every 2 days. Osteoclasts were generated after 8-10 days.
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