The mouse model of acute myocardial infarction (AMI) was performed by permanent ligation of the left anterior descending (LAD) coronary artery. One minute after artery occlusion, the ischemic area was identified and 25 μl of cell suspensions containing 3 × 105 bone-derived Nestin+ cells, Nestin− cells, or saline (vehicle control) was intramyocardially injected into the infarcted border zone as previously described [25 (link)]. Injected cells were labeled with CellTracker CM-Dil (Vybrant™ Dil cell-labeling solution, Thermo Scientific) (50 μg/ml) 30 min prior to transplantation. In the Nestin+ BMSC+AMD3100 treatment group, 5 μl AMD 3100 (200 mmol/L, Selleckchem, Houston, USA) dissolved in saline was intramyocardially injected along with cell treatment. To determine the effect of bone-derived Nestin+ cells and Nestin− cells on infarct size and endogenous CEC recruitment, mouse hearts were harvested 1 week after MI for hematoxylin-and-eosin (HE) and immunofluorescence staining. Hearts were fixed and embedded in paraffin and sectioned into 5-μm slices. Conventional echocardiography (C57BL/6 mice from each group at 1 and 4 weeks after MI) was performed with a mouse echocardiography system (Vevo 2100 Imaging System, VisualSonics, Toronto, Canada), equipped with a 30-MHz phased transducer. Experiments were conducted and data were collected by two individuals in a blinded fashion.
Amd3100
Manufactured by Selleck Chemicals
Sourced in United States, China
AMD3100 is a small molecule that functions as a CXCR4 antagonist. It is commonly used in research applications to inhibit the CXCR4 receptor.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Ly294002, by Abcam (2 mentions)
Ampf str identifier kit, by Thermo Fisher Scientific (2 mentions)
Enz mdv3100, by Selleck Chemicals (2 mentions)
Lncap, by American Type Culture Collection (2 mentions)
Transwell plate, by Corning (2 mentions)
Cisplatin, by Selleck Chemicals (2 mentions)
Recombinant human sdf 1α cxcl12, by R&D Systems (2 mentions)
Cxcl12, by R&D Systems (2 mentions)
Cxcl12, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Merck Group (1 mentions)
Anti phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Rat anti cd31, by Abcam (1 mentions)
Mouse anti ng2, by Merck Group (1 mentions)
Donkey anti rabbit igg conjugated to cy3, by Jackson ImmunoResearch (1 mentions)
Recombinant bmp2, by R&D Systems (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti β actin a1978, by Merck Group (1 mentions)
Azd1208, by Selleck Chemicals (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
34 protocols using amd3100
1
Nestin+ Cell Treatment for Acute Myocardial Infarction
2
Immunofluorescence analysis of bone cells
3
Pretreatment with AMD3100 Assay
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Pretreatment with AMD3100 (Selleckchem, S3013) was performed before the assay. The cells were treated at a concentration of 10 μM in serum-free media (EBM or high glucose DMEM) overnight prior to conducting each assay.
4
Investigating PIM3 and CXCR4 Signaling
5
Latent HIV Provirus Reactivation
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A CD4-untouched pre-selection step was performed to enrich for CD4+ cells. Selected cells were then stimulated in 50% ImmunoCult-XF T cell Expansion Medium (Stem Cell Technologies), 50% conditioned medium, 2.5 μg/mL PHA (Sigma), 5 μM AMD3100 (Selleckchem), 5 μM MVC (Selleckchem), 1 μM EFV (Selleckchem), 5 μM Z-VAD-FMK (Selleckchem), 100 IU IL-2/mL (Miltenyi) for 36-40h to reactivate latent proviruses. HUT4-3 cells, as positive control, were stimulated with PHA 24h prior to staining. For optimized conditions ≥2.5X10E6 Ramos cells (CD19+CD3−) were added as carrier cells. Subsequently, cells were surface stained for immunological characterization or for assessing Env expression. To detect Env, we used 5 μg/mL biotinylated bNAb mix (3BNC117, 10–1074, PG16, 35O22, 10E8 all provided by F. Klein) which was targeted by an Anti-Biotin-VioBright 515 (Miltenyi) secondary antibody. Cells were fixed for 30 min at RT in PBS 2% PFA. Cells were permeabilized twice with ICS Permeabilization Wash buffer (Biolegend) labeled intracellularly with anti-HIV Gag (KC-57PE, Beckman Coulter). ICS labeled samples were washed twice in perm-wash buffer and acquired on an LSRFortessa (BD Bioscience).
6
Fibrocyte Transplantation for Diabetic Wound Healing
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Thirty‐six diabetic rats were randomized into three groups: the NPWT group, NPWT/antagonist group and control group (n = 12/each group). The dorsa of rats were clipped and depilated 24 hrs before the experiment. Rats were anaesthetized with pentobarbitone sodium (40 mg/kg) 5 min. before surgery. Cultured fibrocytes at day 28 were labelled with a membrane‐inserting red dye (PKH‐26, Sigma‐Aldrich) following the manufacturer's protocol. PKH‐labelled cells (7.5 × 105) in 100 μl PBS were injected into the tail veins of SD rats. Immediately, a full‐thickness skin wound (1.5 × 1.5 cm) was created in the mid‐dorsal area of each recipient rat. A medical foam (VSD Medical Technology Inc., Wuhan, China) covered the wound and was exposed to continuous 125 mmHg suction produced by a vacuum‐assisted device in the NPWT group (n = 12). In the control group (n = 12), moist gauze dressings covered the wounds and were changed daily.
In the NPWT/antagonist group (n = 12), PKH‐labelled fibrocytes were incubated in the AMD3100 (CXCR4‐specific antagonist; Selleck) solution of 25 ng/ml concentration in the CO2 incubator for 1 hr and then were centrifuged to obtain pre‐treated fibrocytes. These cells were mixed with PBS, and an equal amount of treated cells (7.5 × 105) in 100 μl PBS were injected into the rat tail veins. Subsequently, wound models and NPWT were performed as described above.
In the NPWT/antagonist group (n = 12), PKH‐labelled fibrocytes were incubated in the AMD3100 (CXCR4‐specific antagonist; Selleck) solution of 25 ng/ml concentration in the CO2 incubator for 1 hr and then were centrifuged to obtain pre‐treated fibrocytes. These cells were mixed with PBS, and an equal amount of treated cells (7.5 × 105) in 100 μl PBS were injected into the rat tail veins. Subsequently, wound models and NPWT were performed as described above.
7
Establishment of Tamoxifen-Resistant Breast Cancer Cell Line
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ER-positive breast cancer T47D cells were purchased from the American Type Culture Collection (ATCC, USA), and 4-hydroxytamoxifen was purchased from Sigma-Aldrich (H7904; USA). To establish the tamoxifen-resistant subline T47DR, we treated T47D cells with 1 μM 4-hydroxytamoxifen continuously and cultured them in RPMI-1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (GIBCO, USA) and 1% penicillin/streptomycin (Beyotime, China) in a humidified condition containing 5% CO2 at 37°C for more than 6 months. After 6 months, the resistance of T47DR cells to tamoxifen was verified by CCK8 assay. AMD3100 was purchased from Selleck (Shanghai, China) and dissolved in phosphate-buffered saline (PBS).
8
Signaling Pathway Protein Analysis
9
Simvastatin Prodrug Activation Protocol
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The simvastatin prodrug was purchased from (Sigma‐Aldrich, St. Louis, MO, USA) and was subjected to activation as described by Sadeghi, et al. 20 Chloro‐methylbenzamido dialkylcarbocyanine (CM‐DiI) was purchased from Invitrogen (Carlsbad, CA, USA). The PI3K inhibitor, LY294002, was purchased from Abcam (Cambridge, MA, USA), and the SDF‐1α/CXCR4 cascade antagonist, AMD 3100, was obtained from Selleck Chemicals (Houston, TX, USA),. SDF‐1α was procured from Proteintech Group (Chicago, IL, USA). The inhibitor and mimic of miR‐9 were synthesised by Bio‐Asia (Jinan, China). The reporter constructs, pGL3‐CXCR4 and pGL3‐mutCXCR4, with a mutated target seed sequence of miR‐9, were also obtained from Bio‐Asia. Real‐time quantitative polymerase chain reaction (PCR) primers were designed by GeneCopia.
10
Validating PCa Cell Lines and CXCR7/CXCR4 Inhibitors
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