Three lots of MSC isolation were performed in accordance with the recommendations of the Ethics Committee of the Institute of Medical Science, the University of Tokyo, Yamaguchi Hospital, and the NTT Medical Center Hospital, after obtaining written informed consent from all subjects in accordance with the Declaration of Helsinki. MSCs were isolated from three individual donors using previously reported methods34 (link). Briefly, the UCs were collected after informed consent was obtained from pregnant women planning to undergo cesarean sections. Frozen-thawed UC tissues were minced into fragments for the improved explant culture method35 (link). The tissue fragments were cultured with α-minimal essential medium (αMEM; Wako) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in the presence of 5% CO2. Fibroblast-like adherent cells that migrated from the UC tissue fragments were harvested using TrypLE Select and were defined as passage 1. These passage 1 MSCs were cryopreserved and transported by air from the Institute of Medical Science, the University of Tokyo, to Karolinska Institutet. The cells were passaged thrice at the Karolinska Institutet, after which, passage 4 MSCs were used for subsequent experimental analyses.
α minimal essential medium αmem
Manufactured by Fujifilm
Sourced in Japan
α-minimal essential medium (αMEM) is a cell culture medium commonly used in laboratory settings. It provides a balanced source of nutrients and supplements to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mc3t3 e1, by Fujifilm (1 mentions)
Penicillin streptomycin, by Fujifilm (1 mentions)
Tgf β2, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Nacalai Tesque (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Penicillin, by Fujifilm (1 mentions)
Streptomycin, by Fujifilm (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Heat inactivated fetal bovine serum, by Cytiva (1 mentions)
Penicillin streptomycin amphotericin b suspension, by Fujifilm (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
49 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq solution, by Takara Bio (1 mentions)
6 protocols using α minimal essential medium αmem
1
Isolation and Expansion of MSCs from UC
2
Culturing Murine Pre-Osteoblast MC3T3-E1 Cells
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The MC3T3-E1 murine pre-osteoblast cell line was purchased from the RIKEN Cell Bank. MC3T3-E1 cells were grown at 37°C in a moist atmosphere of 5% CO2 in α-minimal essential medium (α-MEM, Wako Pure Chemical Co., Osaka, Japan) supplemented with 10%(v/v) fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, USA) and 1%(v/v) penicillin-streptomycin (Wako Pure Chemical, Osaka, Japan).
3
Establishment and Maintenance of Mouse Cell Lines
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Mouse EMRECs were established as described in “Supporting Information ” and maintained in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque). MS1, mouse endothelial cell line, was purchased from the ATCC (Manassas, VA, USA) and maintained in α‐Minimal Essential Medium (αMEM; FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. SAS, human OSCC cell line, was obtained from RIKEN Bioresource Center Cell Bank (Tsukuba, Japan) and maintained in DMEM (Nacalai Tesque) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. TGF‐β2 was purchased from Peprotech (Rocky Hill, NJ, USA).
4
Osteoblast Cell Culture and LPS Treatment
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The MC3T3-E1 mouse calvarial cell line (Riken BioResource Center, Tsukuba, Japan) was used as the osteoblastic cell line. The cells were maintained in α-minimal Essential Medium (α-MEM; FUJIFILM Wako Pure Chemical, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1% Penicillin–Streptomycin–Amphotericin B Suspension (FUJIFILM Wako Pure Chemical) at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. The cells were either treated with 100 ng/mL LPS (ligand of TLR4: E. Coli, L4524, Sigma-Aldrich, St. Louis, MO, USA) or left untreated. The medium was replaced every three days.
5
Osteoclast Differentiation Protocol
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The RAW264.7 cells obtained from Dainippon Pharmaceutical (Osaka, Japan) and maintained in our laboratory [16 (link)] were used as osteoclast precursor cells in this study. Penicillin/streptomycin, bovine serum albumin, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT, USA). Soluble RANKL, α-minimal essential medium (α-MEM), phenol red-free α-MEM/F-12, phosphate-buffered saline (PBS), paraformaldehyde, sucrose, and sodium chloride were purchased from Wako Pure Chemical (Osaka, Japan). NucleoSpin RNA, the RNA PCR Kit (PrimeScript), and SYBR Premix Ex Taq solution were purchased from Takara Bio (Otsu, Japan). Alexa Fluor 488-phalloidin and 49,6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific (Rockford, IL, USA). The TRAP staining kit was purchased from Cosmo Bio (Sapporo, Japan). The mouse anti-carbonic anhydrase II (sc-48351), mouse anti-matrix metalloproteinase-9 (sc-393859), mouse anti-cathepsin K (sc-48353), and mouse anti-β-tubulin (sc-5274) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Biotin-conjugated goat anti-mouse antibodies were obtained from Abcam (Cambridge, UK). The western ECL substrate kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA).
6
Isolation and Expansion of Mesenchymal Stem Cells from Umbilical Cords
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Three lots of MSC isolation were performed in accordance with the recommendations of the Ethics Committee of the Institute of Medical Science, the University of Tokyo, Yamaguchi Hospital, and the NTT Medical Center Hospital, after obtaining written informed consent from all subjects in accordance with the Declaration of Helsinki. MSCs were isolated from three individual donors using previously reported methods (15) . Brie y, the UCs were collected after informed consent was obtained from pregnant women planning to undergo cesarean sections. Frozen-thawed UC tissues were minced into fragments for the improved explant culture method (16). The tissue fragments were cultured with α-minimal essential medium (αMEM; Wako) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in the presence of 5% CO 2 . Fibroblast-like adherent cells that migrated from the UC tissue fragments were harvested using TrypLE Select and were de ned as passage 1. These passage 1 MSCs were cryopreserved and transported by air from the Institute of Medical Science, the University of Tokyo, to Karolinska Institutet. The cells were passaged thrice at the Karolinska Institutet, after which, passage 4 MSCs were used for subsequent experimental analyses.
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