We assessed cell migration in 12-well Transwell plates (Corning Inc.) with filters with 8-μm pores. In brief, we seeded 1 × 104/well MSCs or ECs in the upper chambers and preincubated them with vehicle, 20 μM Cyclo(-RGDfK) (an integrin αvβ3 inhibitor; MedChemExpress), 20 μM AMD 3100 (a CXCR4 inhibitor; MedChemExpress), 20 μM ATN-161 (an integrin α5β1 inhibitor; Selleck Chem), 1 μM J-113863 (a CCR1 inhibitor; MedChemExpress), 1 μM CCR3 antagonist 1 (a CCR3 inhibitor; MedChemExpress), 1 μM TAK-220 (a CCR5 inhibitor; MedChemExpress), 20 μM Ki8751 (a VEGFR2 inhibitor; MedChemExpress), 10 μM SB225002 (a CXCR2 inhibitor; MedChemExpress) or 2 μM Y15 (a FAK inhibitor; Sigma-Aldrich) for 1 h. Then, we incubated them with medium in the lower chambers for an additional 24 h with the inhibitor or vehicle remaining in the upper chambers. At the end of incubation, the cells were fixed with 10% formaldehyde for 30 min. The cells on the lower surface were stained with crystal violet (Sigma-Aldrich) and quantified by counting five random fields per well using a microscope (Olympus) at 200 × magnification.
Amd3100
Manufactured by MedChemExpress
Sourced in United States, China
AMD3100 is a small molecule that functions as a CXCR4 antagonist. It is commonly used in research applications as a tool compound to study the CXCR4 receptor and its biological role.
Automatically generated - may contain errors
Lab products found in correlation
Cxcl12, by Thermo Fisher Scientific (3 mentions)
Sb225002, by MedChemExpress (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Crystal violet, by Merck Group (1 mentions)
Cyclo rgdfk, by MedChemExpress (1 mentions)
Atn 161, by Selleck Chemicals (1 mentions)
J 113863, by MedChemExpress (1 mentions)
Ki8751, by MedChemExpress (1 mentions)
Wz811, by MedChemExpress (1 mentions)
Amd070, by MedChemExpress (1 mentions)
Msx 122, by MedChemExpress (1 mentions)
Transwell chamber, by Corning (1 mentions)
Sdf 1, by Thermo Fisher Scientific (1 mentions)
Enzyme free cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Trypsin solution, by Thermo Fisher Scientific (1 mentions)
Hbss with ca2 and mg2, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Linear 25 kda polyethylenimine pei, by Polysciences (1 mentions)
White low volume 384 well plates, by Corning (1 mentions)
Sch772984, by MedChemExpress (1 mentions)
15 protocols using amd3100
1
Cell Migration Inhibition Assay
2
Targeting CXCR4 in Leukemia Therapy
3
Transwell Migration Assay for MSCs
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Transwell chambers (Corning, New York, USA) with a 6.5 mm diameter and a polyethylene terephthalate (PET) track-etched membrane with 8.0 μm pores were used in the Transwell migration assay. 1 × 104 cells were seeded in the upper chambers in 200 μL of DMEM/F-12 medium containing 0.1% (g/mL) BSA, and 600 μL DMEM/F-12 culture medium supplemented with 10% (v/v) FBS was added in the lower chambers. For the chemotaxis assay, SDF-1 (PeproTech, Rocky Hill, NJ, USA) at concentrations of 0, 10, 20, 50, and 100 ng/mL were used as the lower chamber medium. In the chemotaxis inhibition assay, MSCs and Pre-MSCs were incubated with 10 μg/mL AMD3100 (MedChemExpress, Shanghai, China), an antagonist of CXCR4 for 2 h before seeded in the upper chambers. The cells without preincubation were used as control. The concentration of SDF-1 in the lower chambers was 100 ng/mL. After 24 h culture, cells in the upper chambers were removed and the membranes were fixed with 4% paraformaldehyde for 20 min. Then, the cells migrated to the lower side of the filter were stained with 0.5% crystal violet for 10 min. At last, the cells were observed with the microscope, and 5 microimages with 100x magnification were taken randomly to analyze the migrated cells.
4
Chemokine Receptor Binding Assay
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HBSS (with Ca2+ and Mg2+), Dulbecco’s Modified Eagle’s Medium (DMEM; high glucose), 0.05% trypsin solution and penicillin/streptomycin solution, and enzyme-free cell dissociation buffer were purchased from ThermoFisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from Bodinco (Alkmaar, the Netherlands). Linear 25 kDa polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA, USA). Bovine serum albumin (BSA) was obtained from Melford (Ipswich, UK). Furimazine (N1130) was purchased from Promega (Madison, WI, USA). The 96-well white and black cell culture plates used were obtained from Greiner Bio-one (Kremsmünster, Austria). White low-volume 384-well plates were bought from Corning (Corning, NY, USA). Human recombinant CXCL12, CXCL10, and the fluorescently labeled chemokines CXCL12-AZD488, CXCL12-AZD546, CXCL12-AZD594, CXCL12-AZD647, and CXCL10-AZD488 were purchased from Protein Foundry (Milwaukee, WI, USA). IT1t (Bio-Techne Ireland Limited, Dublin, Ireland), AMD3100 and Burixafor were purchased from MedChemExpress (Princeton, NJ, USA), and VUF25444, VUF15485, VUF25550 and VUF16545 were synthetized in-house.
5
Signaling Pathway Inhibitor Protocol
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The CXCR4 inhibitor AMD3100 (HY-10046), ERK inhibitor SCH772984 (HY-50846), PI3K inhibitor LY294002 (HY-10108), mTOR inhibitor rapamycin (HY-10219), PKC inhibitor GO6983 (HY-13689) and PKA inhibitor H89 dihydrochloride (HY-15979A) were purchased from MedChemExpress (USA). Recombinant human CXCL12 protein was purchased from Bio-Techne (350-NS-010, R&D Systems, MN, USA). All the agents were used according to the manufacturer's instructions.
Detailed descriptions of all other materials and methods can be found in the onlineSupplementary Materials .
Detailed descriptions of all other materials and methods can be found in the online
6
Isolation and Expansion of Rabbit PB-MSCs
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The Animal Care and Use Committee of Peking University Third Hospital approved all of the protocols of animal experiments which were implemented follow the Guide for the Care and Use of Laboratory Animals. Peripheral blood (PB, 20 mL) was isolated from the central auricular arteries of New Zealand White rabbits after mobilizing with granulocyte colony stimulating factor (G-CSF, Qilu Pharmaceutical Co. Ltd.) and AMD3100 (MedChemExpress LLC., USA). Peripheral blood mononuclear cells (MNCs) were collected by using the method of density gradient centrifugation, and cultured in medium of α-MEM with 15% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin. Culture medium was replaced every 3 days until the confluence of primary PB-MSCs reached around 90%, and then subculture was carried out at ratio of 1:3. PB-MSCs at passage 3 [PB-MSCs (P3)] were used for subsequent experiments (Fu et al., 2011 (link)).
7
Biochemical Pathway Modulation Protocol
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Vincristine sulfate, which was product of Taoshu Biotechnology (Shanghai, China), was prepared in phosphate buffer solution (PBS). AMD3100 and PD98059 from MedChemExpress (Shanghai, China) were formulated separately in dimethyl sulfoxide (DMSO) and anhydrous ethanol. The AKT, ERK, phospho-AKT and phospho-ERK antibodies were procured from Cell Signaling Technology (Danvers, MA, USA)., while the CXCR4 and IFI6 antibodies were procured separately from Solibao Biotechnology (Beijing, China) and ImmunoWay Biotechnology (Plano, TX, USA). Proteintech Group (Wuhan, China) was the provider of the secondary antibody used herein for Western blot.
8
Inflammatory Stromal Fibroblasts Modulate Epithelial Responses
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The effects of NFs and INFs on epithelial cells were investigated through an indirect co-culture model by using conditioned medium (CM). In brief, NFs or INFs were rinsed with PBS. Then fresh serum-free DMEM/F12 medium was added to the culture dishes and cultured for another 24 h. Cell culture supernatant was collected and used as CM by mixture with complete medium at a 9:1 ratio. Finally, cell culture supernatants were collected and epithelial cells were harvested for subsequent analysis after 3-day culture in CM. Epithelial cells cultured in MSM were taken as control.
To investigate whether inflammatory stromal fibroblasts induced by LPS or LTA could promote SDF-1-mediated inflammatory response in epithelial cells, epithelial cells were cultured in CM from LPS- or LTA-treated NFs. Firstly, CM was collected from NFs that were pre-treated by LPS or LTA as aforementioned. And epithelial cells were pretreated by AMD3100 (MedChem Express, Princeton, NJ), a specific inhibitor of the receptor of SDF-1, for 24 h at the concentration of 25 μg/ml. Then, AMD3100-pre-treated and untreated epithelial cells were cultured in CM from LPS- or LTA-treated NFs. Finally, cell culture supernatants were collected to measure the secretion TNF-α at day 3 after culture using an ELISA Kit. Epithelial cells cultured in MSM were used as control.
To investigate whether inflammatory stromal fibroblasts induced by LPS or LTA could promote SDF-1-mediated inflammatory response in epithelial cells, epithelial cells were cultured in CM from LPS- or LTA-treated NFs. Firstly, CM was collected from NFs that were pre-treated by LPS or LTA as aforementioned. And epithelial cells were pretreated by AMD3100 (MedChem Express, Princeton, NJ), a specific inhibitor of the receptor of SDF-1, for 24 h at the concentration of 25 μg/ml. Then, AMD3100-pre-treated and untreated epithelial cells were cultured in CM from LPS- or LTA-treated NFs. Finally, cell culture supernatants were collected to measure the secretion TNF-α at day 3 after culture using an ELISA Kit. Epithelial cells cultured in MSM were used as control.
9
Cell Culture and Compound Treatments
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HCC cell lines SMMC-7721, Huh7, HepG2, Hep3B and 293T cells were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HCC Cell lines were routinely cultured in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). Spheres from OV6+ HCC cells were cultured in DMEM/F-12 (Gibco) without FBS, but with supplement of ITS Liquid Media (Sigma-Aldrich, St. Louis, MO, USA), B27 (Invitrogen, Carlsbad, CA, USA), EGF Recombinant Human Protein Solution (Gibco), FGF-Basic (AA 1-156). Recombinant CXCL12 protein (R&D Systems, Minneapolis, MN, USA) was added in conditional media for the CXCL12/CXCR4 related assays. AMD3100 (Plerixafor, HY-10046) was purchased from MedChem Express (Monmouth Junction, NJ, USA). MG132 (M8699) and CHX (R750107) were obtained from Sigma-Aldrich.
10
Chemotaxis Assay for MDSCs
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MDSCs were divided into four treatment groups by coculturing with PBS, exosomes, exosomes plus AMD 3100 (MedChemExpress, USA) of 10 μg/500 μl, and AMD 3100 for 24 h, respectively. Then, MDSCs were harvested, adjusted to a concentration of 5 × 105/200 μl RPMI 1640, added into the upper chambers, and divided into four different treatment groups: PBS, exosomes, exosomes plus AMD3100, and AMD3100. CXCL12 was added in the lower chamber and cocultured for 24 h. 500 μl RPMI 1640 with 100 ng CXCL12 (Abcam, USA) was added into the lower chambers and then incubated at 37°C for 24 h, according to Liu et al. [25 (link)]. The cells migrated to the lower surface had been fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 10 min. Cells were counted and averaged through the selection of 5 random fields/well under a light microscope (Nikon, Japan).
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