ANCs and ReANCs were functionalized with AMD3100 as previously described (Zevon et al., 2015 (link); Kantamneni et al., 2017 (link)). Briefly, AMD3100 (EMD Millipore, Burlington, MA) was dissolved in sterile water at a concentration of 1.25 μM and adsorbed onto nanoparticles by adding 388 μL of the AMD3100 solution to 3.5 mL of ANCs or ReANCs (1x concentration, i.e., total albumin concentration of 5 mg/mL), followed by constant agitation at room temperature for 3–4 h. This concentration of AMD3100 was found to be the optimal loading condition based on previously described in vitro experiments (Zevon et al., 2015 (link)). For particles that were dual loaded with AMD3100 and Dox (Dox-fANCs or Dox-fReANCs), AMD3100 functionalization was performed prior to Dox loading.
Amd3100
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, Italy, Sao Tome and Principe, Morocco, Macao, Japan, Canada
AMD3100 is a small molecule that acts as a CXCR4 antagonist. It is used as a laboratory tool in research applications.
Automatically generated - may contain errors
Lab products found in correlation
G csf, by Amgen (10 mentions)
Sdf 1, by R&D Systems (8 mentions)
Sdf 1, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Pharm lyse buffer, by BD (7 mentions)
Cxcl12, by Thermo Fisher Scientific (7 mentions)
Trypsin, by Thermo Fisher Scientific (6 mentions)
Sdf 1, by Thermo Fisher Scientific (6 mentions)
Ly294002, by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Cxcl12, by R&D Systems (5 mentions)
U0126, by Merck Group (5 mentions)
Sdf 1α, by Merck Group (5 mentions)
Matrigel, by BD (5 mentions)
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Sdf 1α, by Thermo Fisher Scientific (4 mentions)
Pd98059, by Merck Group (4 mentions)
U0126, by Cell Signaling Technology (4 mentions)
Recombinant human cxcl12, by Thermo Fisher Scientific (4 mentions)
328 protocols using amd3100
1
Nanoparticle Functionalization with AMD3100
2
Stem Cell Therapy for Parkinson's Disease
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PD rats were randomly divided into three groups: NSC-grafted group (n = 12), NSC-grafted + AMD3100 group (n = 12), and AMD3100 group (n = 12). The untreated rats received saline instead of 6-OHDA as the control group (n = 12). The rats in the sham group (n = 12) received serum-free Dulbecco’s modified Eagle’s medium instead of NSCs and AMD3100. For the NSC-grafted group and the NSC-grafted + AMD3100 group, the NSC suspension was incubated in serum-free Dulbecco’s modified Eagle’s medium, and 5 μL of NSC suspension (1.5 × 104 cells/μL) were injected into the right substantia nigra of PD rats at the following coordinates: 5 μL at anterior–posterior, –3.6 mm; lateral, –2.0 mm; vertical, –8.5 mm; and 5 μL at anterior–posterior, +0.7 mm; lateral, –3.0 mm; and vertical, –5 mm from bregma. The injection speed was 1 μL/minute, and the needle was maintained in place for 15 minutes. The incisions were sutured and rats were intramuscularly injected with 100 kU of penicillin to prevent infection. The rats in the sham and AMD3100 groups received serum-free Dulbecco’s modified Eagle’s medium instead of cell suspensions. AMD3100 (Sigma-Aldrich Corp.) was dissolved in 1 mg/mL of sterile saline and intraperitoneally administered at a dose of 1.25 mL/kg once daily for 3 days (Saha et al., 2013) in the NSC-grafted + AMD3100 and AMD3100 groups.
3
Measuring T Cell Chemotaxis Towards CCL21 and CXCL12
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The assay was performed and analyzed as described [30] (link). Briefly, cells from the afferent lymph of sheep or mouse mononuclear splenocytes were incubated in RPMI containing 0.5% bovine serum albumin (Invitrogen) for 1 h. 5–10×105 cells in 100 µl were added to 5-µm-pore-sized, 24-well tissue culture inserts (Corning costar). Recombinant mouse CCL21 (R&D Systems) and recombinant human or mouse CXCL12 (R&D Systems) were titrated in triplicates, and cells were allowed to migrate for 1.5 h. Lymphocytes in the migrated and input wells were quantified using a bead standard (15 µm polystyrene beads, Polyscience Inc.) combined with the flow cytometric analysis of T cell subsets.
To test the effect of AMD3100 on T cell chemotaxis, mouse splenocytes were incubated with 5 or 25 µM AMD3100 (Sigma-Aldrich) for 15–30 min in RPMI1640 with 0.5% BSA, before subjecting them to a chemotaxis assay in the presence of AMD3100.
To test the effect of AMD3100 on T cell chemotaxis, mouse splenocytes were incubated with 5 or 25 µM AMD3100 (Sigma-Aldrich) for 15–30 min in RPMI1640 with 0.5% BSA, before subjecting them to a chemotaxis assay in the presence of AMD3100.
4
Genetic Mouse Models of Pulmonary Hypertension
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Egln1Tie2Cre, Egln1/Cxcl12Tie2Cre mice were generated as described previously [7 (link)]. Foxm1 floxed mice [11 (link)] were bred with Egln1Tie2Cre mice to generate Egln1/Foxm1Tie2Cre mice. For CXCR4 inhibitor AMD3100 treatment study, Egln1Tie2Cre mice at the age of 3 weeks were treated with vehicle (PBS) or CXCR4 inhibitor AMD3100 (7.5 mg/kg, daily) (MilliporeSigma, St. Louis, MO, USA) for 5 weeks. Right ventricular systolic pressure (RVSP) in mice was measured as described previously [7 (link),9 (link),35 (link)]. The experiments were conducted according to the National Institutes of Health guidelines on the use of laboratory animals. The animal care and study protocol were approved by the Institutional Animal Care and Use Committee of Northwestern University (#IS00006960, approved date 15 September 2017) and the University of Arizona (#19-513, approved date 15 August 2019).
5
AMD3100 Inhibits Lung Cancer Metastasis
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6
Transwell Migration Assay for MSCs
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The test groups for the transwell migration assay were the same as described above, except for the deletion of the hypoxia supernatant group and infarcted MTE group. In addition, AMD3100, an inhibitor of SDF-1/CXCR4 axis, was used in the migration assay. Briefly, MSCs were harvested, resuspended in serum-poor medium, and seeded into the upper chamber (100 μL, 1 × 106/mL) of a 24-well transwell plate (Corning Costar, USA) with 8-μm pore filters. In the groups requiring US treatment, human MSCs were seeded into 6-well plates first, subjected to UTMD, and then seeded into the upper chamber. In the group containing AMD3100, MSCs were pretreated by coculturing with AMD3100 (5 μg/mL) (Sigma) for 30 min. The supernatants or myocardial tissue extracts assigned to the different groups were placed into each lower chamber (500 μL). Following a 5 h incubation, the cells that did not migrate from the upper chamber were removed with a cotton swab. The cells on the lower surface of the membrane were stained with 0.1% (w/v) crystal violet solution for 20 min, and the number of them was calculated under a light microscope at 200x magnification in five randomly selected fields. Each experiment was repeated three times in duplicate.
7
Adoptive B Cell Immunotherapy for Metastatic Breast Cancer
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Healthy Balb/c mice were inoculated with 5×104 4T1 cells into the mammary fat pad to induce spontaneous pulmonary metastases. Fourteen days after tumor inoculation, the tumor- bearing mice were treated with tail vein injection of activated 4T1 TDLN B cells. Commencing on the day of the effector B cell transfer, intraperitoneal (i.p.) injections of IL-2 (40,000 IU) (Prometheus Laboratories, San Diego, CA) were administered in 0.5 ml of PBS and continued twice daily for 8 doses. About 2 weeks after B cell transfer, all mice were sacrificed and lungs were harvested for enumeration of spontaneous pulmonary metastatic nodules. At the same time, spleens were collected for purification of splenic T and B cells as described above.
For 4T1 TDLN B cell cytotoxicity in the presence of anti-FasL antibody (Biolegend Inc., San Diego, CA) and/or AMD3100 (Sigma, Atlanta, GA), the effector B cells were generated as described above and cultured with 4T1 cells with the admixture of 20 μg /ml anti-FasL and/or 2.5 μg /ml AMD3100 to block FasL and/or CXCR4; respectively.
For 4T1 TDLN B cell cytotoxicity in the presence of anti-FasL antibody (Biolegend Inc., San Diego, CA) and/or AMD3100 (Sigma, Atlanta, GA), the effector B cells were generated as described above and cultured with 4T1 cells with the admixture of 20 μg /ml anti-FasL and/or 2.5 μg /ml AMD3100 to block FasL and/or CXCR4; respectively.
8
AMD3100 Subcutaneous Wound Injection
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9
Breast Cancer Xenograft Model in Mice
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Balb/c nu/nu mice (3–4 weeks old) were purchased from Beijing Huafukang Bioscience Co., Inc., and Balb/c mice (6–8 weeks old) were purchased from Liaoning Changsheng Biotechnology Co., Ltd., in China and raised in the SPF animal laboratory. All animal experiments were conducted in accordance with the recommendations of the Policy on the Humane Care and Use of Laboratory Animals and approved by the Ethics Committee of HUST. Then, 50 μl of a MDA-MB-231 cell suspension (5×106) with 50 μl of Matrigel and 100 μl of a 4T1 cell suspension (1×106) were injected into the fourth left mammary fat pad. Tumor volume was calculated by the formula length×width2×0.5. The mice were randomly subdivided into five groups (8 mice/group): (1) the control group: saline 150 μl i.g.; (2) the losartan group: losartan 150 μl of 40 mg/kg/d i.g. (3) the mock group: MOCK cells+saline i.p.; (4) the AGTR1high group: AGTR1high cells+saline i.p.; and (5) the AGTR1high+AMD3100 group: AGTR1high cells+AMD3100 (2.5 mg/kg/d, Sigma, St. Louis, MO, USA) i.p. After two weeks (MDA-MB-231 tumors) or one week (4T1 tumors) of tumor implantation, saline or losartan were given to the mice orally for consecutive days. The mice were sacrificed when first tumor volume reached 2000 mm3.
10
Optimizing Ibrutinib and Doxorubicin Combination
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The optimal time and dose of ibrutinib (IBR) and doxorubicin (DOX) on Ri-1 and Raji cell lines have been previously investigated [19 (link)]. The ibrutinib IC50 for inhibition of Ri-1 and Raji cells proliferation was 0.514 μM and 6.18 μM, respectively, while the values of IC50 for DOX were 0.833 μM and 0.174 μM for Ri-1 and Raji cell lines, respectively.
The concentration of AMD3100 was chosen following Azab et al. [24 (link)]. DOX, IBR, and AMD3100 (plerixafor) were purchased from Sigma-Aldrich (Darmstadt, Germany). Stock concentrations for DOX (1 mM) and AMD3100 (5 mM) were made in water and stored at -20 °C, while IBR (10 mM) was dissolved in DMSO (Sigma Aldrich) and stored at 4 °C. Working stocks were made in culturing media. Ri-1 and Raji cells (0.5 × 106) were grown in 1 mL of the medium supplemented with 1. AMD 3100 (50 μM), DOX (0.05 μg/mL), and IBR (0.4 μM) alone, or 2. DOX (0.05 μg/mL) with AMD-3100 (50 μM) and IBR (0.4 μM) with AMD3100 (50 μM) under standard conditions for 48 h. Appropriate untreated controls were prepared. Cells were washed in PBS to remove compounds, and cell viability was assessed by trypan blue staining in an automated cell counter and immediately analyzed with optical tweezers. Experiments were repeated twice for both cell lines and all of the tested drugs.
The concentration of AMD3100 was chosen following Azab et al. [24 (link)]. DOX, IBR, and AMD3100 (plerixafor) were purchased from Sigma-Aldrich (Darmstadt, Germany). Stock concentrations for DOX (1 mM) and AMD3100 (5 mM) were made in water and stored at -20 °C, while IBR (10 mM) was dissolved in DMSO (Sigma Aldrich) and stored at 4 °C. Working stocks were made in culturing media. Ri-1 and Raji cells (0.5 × 106) were grown in 1 mL of the medium supplemented with 1. AMD 3100 (50 μM), DOX (0.05 μg/mL), and IBR (0.4 μM) alone, or 2. DOX (0.05 μg/mL) with AMD-3100 (50 μM) and IBR (0.4 μM) with AMD3100 (50 μM) under standard conditions for 48 h. Appropriate untreated controls were prepared. Cells were washed in PBS to remove compounds, and cell viability was assessed by trypan blue staining in an automated cell counter and immediately analyzed with optical tweezers. Experiments were repeated twice for both cell lines and all of the tested drugs.
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