Human mesenchymal stem cells (hMSCs) were isolated from human bone marrow provided by commercial sources (Cyagen Biosciences). Briefly, cells were obtained from donors by bone marrow aspiration, then monocyte density centrifugation was performed and selected for adherent culture. Standard analytical methods were used to screen cell growth and differentiation into fat and bone. The cells from five donors were mixed in reserve before performing all the experiments in this manuscript. The age and sex of the five hMSCs donors were 36 years (male), 38 years (female), 38 years (female), 41 years (female), and 42 years (male), respectively. hMSCs were cultured in growth media (Cyagen, HUXMA-90011), except as noted. For osteogenic differentiation studies, cells were cultured in an osteogenic medium (Cyagen, HUXMA-90021) to assay the hMSCs osteogenic differentiation capability.
Huxma 90011
Manufactured by Cyagen
Sourced in United States, China
The HUXMA-90011 is a lab equipment product manufactured by Cyagen. It is designed for performing various laboratory tasks, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information may be available from the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Huxma 90021, by Cyagen (2 mentions)
Glutamine, by Cyagen (2 mentions)
Penicillin streptomycin, by Cyagen (2 mentions)
Huxma 01001, by Cyagen (2 mentions)
Huxmd 90011, by Cyagen (2 mentions)
Cd105, by Abcam (2 mentions)
Huxuc 90011, by Cyagen (2 mentions)
Cd11b, by Abcam (2 mentions)
Hek293 cell line, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Cyagen (1 mentions)
Trypsin without edta, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Huxma 90031, by Cyagen (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Cell Applications (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Human bmscs, by American Type Culture Collection (1 mentions)
15 protocols using huxma 90011
1
Isolation and Characterization of Human Mesenchymal Stem Cells
2
Isolation of Human Bone Marrow Mesenchymal Stem Cells
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Human (h)BMSCs from healthy volunteers were extracted from bone marrow as described in our previous research (24 (link)). In brief, volunteers were recruited from patients undergoing bone marrow aspiration at Xiangya Hospital (Changsha, Hunan, China) from January 2015 to December 2016. Volunteers with immune system diseases and blood test abnormal were excluded. A total 8 patients (3 male and 5 female) from age 22–45 years old (mean age 36 years) were recruited. Bone marrow aspiration were performed to acquire the bone marrow. Ficoll density gradient media (density 1.077 g/cm3; GE Healthcare) method was using to isolate hBMSC. Buffy coats, which including hBMSCs, were collected after centrifugation (1,100 × g; 20 min at 37°C). After isolation from the bone marrow aspirates, hBMSCs were incubated in a complete hBMSC medium supplemented with 10% fetal bovine serum (FBS; HUXMA-90011; Cyagen Biosciences, Inc.), 100 U/ml penicillin/streptomycin and glutamine at a density of 107 cells per 100 mm dishes at 37°C in a 5% CO2 incubator. The third generation of the cells was subjected to subsequent experiments. The HEK293 cell line (Thermo Fisher Scientific, Inc.) was cultured in high-glucose (4,500 mg/l) DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator.
3
Expanding Human Bone Marrow Stromal Cells
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Briefly, human BMSCs (passage 1) were purchased from 307-Ivy Translation Medicine Center (Beijing, China) and grown in hMSC basal media (HUXMA-90011, Cyagen, USA) supplemented with 10% fetal bovine serum (Cyagen, USA) and 1% penicillin-streptomycin (Cyagen, USA) and glutamine (Cyagen, USA) at 37°C with 5% CO2 and saturated humidity. We used 0.25% trypsin without EDTA (Solarbio Science & Technology Co. Ltd., Beijing, China) to digest cells when the cells had grown to a confluence of 70–80% in the flask (431464U, Corning, USA). The media were changed every two days, and cells at passage 3–5 were used for the following experiments. The BMSC phenotypes are identified (S Figure 2 ).
4
Human Glioma and Astrocyte Cell Lines
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Human glioma cell lines (U87MG and A172) and normal human astrocytes (NHAs) were obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific; USA) with 10% fetal bovine serum (FBS; Gibco; USA). Human bone marrow-derived MSCs were purchased from Cyagen (Suzhou, China) and cultured in mesenchymal stem cell complete medium (HUXMA-90011; Cyagen; China). These cell lines were maintained in a humidified chamber containing 5% CO2 at 37° C.
5
Differentiation of Bone Marrow Stem Cells
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BMSCs (3 × 104/cm2) were cultured in complete growth medium (HUXMA-90011, Cyagen Biosciences) and incubated at 37°C under 5% CO2. For osteogenic differentiation, the cells were subsequently cultured in osteogenic induction medium (HUXMA-90021; Cyagen Biosciences). For adipogenic differentiation of BMSCs, cells were induced in adipogenic induction medium (HUXMA-90031; Cyagen Biosciences). The cells were maintained by the addition of fresh osteogenic induction medium every 2–3 days.
6
Co-culture of bMSCs and HUVECs for LPS-stimulation
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Commercialized primary human bMSCs (HUXMA-01001, Cyagen, China) were cultured in a growth medium for human MSCs (HUXMA-90011, Cyagen, China), and cells at passages 3–6 were used for experiments. Human umbilical vein endothelial cell lines (HUVECs, Cell Applications, USA) were cultured in DMEM/F12 medium (Gibco, USA) in an environment of the humidified atmosphere at 37 °C with 5% CO2. All culture mediums contained 10% EV-free FBS (Exo-FBS-50a-1, BI, USA) with 1% penicillin–streptomycin (Gibco, USA). For co-culture experiments, bMSCs were seeded on Transwell inserts in six-well plates (3450, Corning, USA) and HUVECs were seeded in six-well plates. When HUVECs attained 80% confluency, bMSCs and HUVECs were co-cultured in a refreshing medium. HUVECs were stimulated with LPS (1 μg/mL) for 6 h and then were collected for related experiments.
7
Expansion and Characterization of Human BMSCs
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Human BMSCs were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). BMSCs were cultured in basal medium (HUXMA-90011, Cyagen, Santa Clara, CA, USA) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and glutamine at 37 °C in saturated humidity at 5% CO2. Early passage BMSCs (passages 3–5) were used for the in vitro study. The human umbilical vein endothelial cells (HUVECs), human skin fibroblasts (HSFs) and HEK-293 cells used in our present study were obtained from the cell bank of the Chinese Academy of Medical Sciences (Beijing, China). HUVECs and HSFs were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco BRL, Grand Island, USA) with 10% FBS and 1% penicillin-streptomycin. All cells were passaged every 2–3 days when they reached approximately 90% confluence.
8
Culture and Maintenance of Human Cell Lines
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Human BMSCs (ATCC; Manassas, VA, USA) were cultured in basal medium (HUXMA-90011, Cyagen Biosciences, Santa Clara, CA, USA) containing 10% fetal bovine serum (FBS). HUVEC and HEK-293 cells were obtained from the cell bank of the Chinese Academy of Medical Sciences (Beijing, China) and cultured in high-glucose Dulbecco's modified Eagle’s medium (DMEM, Gibco BRL, Grand Island, NY, USA) containing 10% FBS and 1% penicillin–streptomycin.
9
Isolation and Culture of Human BMSCs
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Human BMSC was isolated from bone marrow when the healthy fractured patients underwent internal fixation surgery. The recruited patients were young adults who suffered from accidental trauma, without basic diseases and long-term drug administration before. Then the BMSC within the bone marrow was obtained and purified by serial passages.78 (link) Bone-marrow aspirates from humans were cultured in a complete medium (Cyagen, HUXMA-90011) to obtain adherent cells (90% confluency, P0 cells). They were replated at a low density (70 cells per cm−2) to obtain P1 cells (70% confluency). The P1 cells could be divided and preserved using liquid nitrogen. Those cells were thawed and cultured with a high density and then replated at a low density to obtain P2 cells, which were used to perform the following experiments.
10
Osteogenic Differentiation of Human and Mouse Bone Marrow Stromal Cells
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HBMSCs and mBMSCs were provided by Cyagen Biosciences (HUXMA-01001, MUBMX-01001, Guangzhou, China), which can differentiate into osteoblasts, chondrocytes, and adipocytes under specific inductive conditions. Adherent hBMSCs were incubated in culture flasks in a special complete growth medium (HUXMA-90011, Cyagen Biosciences, Inc., Guangzhou, China) in a cell incubator at 37 °C with 5% CO2 and were passaged at nearly 80–90% confluence. Cells from passages two to six were used in subsequent experiments.
Specific antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and FOXA1 were purchased from PROTEINTECH (Chicago, USA). Runt-related transcription factor 2 (RUNX2), extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2) were obtained from Cell Signalling Technology (Danvers, MA, USA). Specific antibodies against collagen type I alpha 1 (COL1A1) and Osterix (SP7) were purchased from Abcam (Cambridge, UK) and PROTEINTECH (Chicago, USA). A phospho-p44/42 MAPK (P-ERK1/2) inhibitor, PD98059, was purchased from MedChemExpress (New Jersey, USA).
Specific antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and FOXA1 were purchased from PROTEINTECH (Chicago, USA). Runt-related transcription factor 2 (RUNX2), extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2) were obtained from Cell Signalling Technology (Danvers, MA, USA). Specific antibodies against collagen type I alpha 1 (COL1A1) and Osterix (SP7) were purchased from Abcam (Cambridge, UK) and PROTEINTECH (Chicago, USA). A phospho-p44/42 MAPK (P-ERK1/2) inhibitor, PD98059, was purchased from MedChemExpress (New Jersey, USA).
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