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).
Cxcl12
Manufactured by Abcam
Sourced in United States
CXCL12 is a chemokine that plays a role in the regulation of cell migration and adhesion. It is involved in various physiological and pathological processes, including immune response, stem cell homing, and cancer metastasis.
Automatically generated - may contain errors
Lab products found in correlation
Cxcr4, by Abcam (4 mentions)
Amd3100, by MedChemExpress (1 mentions)
Jc 1 fluorescent probe, by Beyotime (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Col1a1, by Abcam (1 mentions)
Tunel detection kit, by Roche (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Eclipse 80i light microscope, by Nikon (1 mentions)
Transwell insert, by Merck Group (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Goat anti mouse or anti rabbit secondary antibody, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
16 protocols using cxcl12
1
Chemotaxis Assay for MDSCs
2
Molecular Profiling of Cardiac Tissues
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Antibodies for ACTN2, ANK2, ATXN1, COL1A1, COL4A4, COL6A2, CXCL12, SNCA, VDR, WFS1 were purchased from Abcam (Cambridge, MA, USA). The genotyping primers of these genes were listed in Supplementary Table 1. GAPDH was used as interior references for myocardial tissues, which were also purchased from Abcam (Cambridge, MA, USA). The ROS fluorescent probes and JC-1 fluorescent probes were purchased from Beyotime Biotechnology (Shanghai, CHINA). MitoTracker Deep Red was purchased from Invitrogen (Carlsbad, CA, USA). The TUNEL detection kit was purchased from Roche (Indianapolis, IN, USA). All other chemicals were purchased from Sigma unless specifically mentioned otherwise.
3
Western Blot Analysis of EPCs
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Proteins were extracted separately from EPCs, GFP-EPCs, and CXCL12-EPCs with RIPA (Millipore, Temecula, CA, USA). The western blot protocol was carried out as described previously [36 (link)]. The primary antibodies were CXCL12 and VEGF (1:1000; Abcam, Cambridge, MA, USA), and β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
4
Immunohistochemical Staining of CXCR4 and CXCL12
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Immunohistochemical staining was performed as previously described (6 (link)). The primary antibodies used were as follows: Anti-NE (1:200; cat. no. orb1614; Biorbyt Ltd., Cambridge, UK), anti-C-X-C chemokine receptor type 4 (CXCR4; 1:50; cat. no. ab124824, Abcam) and anti-C-X-C motif chemokine 12 (CXCL12; 1:50; cat. no. sc-28876; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Samples were then incubated with the anti-rabbit (1:2,000; cat. no. ab97051; Abcam) secondary antibody for 1 h at room temperature under gentle agitation. Samples were subsequently visualized under an Eclipse 80i light microscope (Nikon Corporation, Tokyo, Japan) and representative images were captured for analysis.
5
Immunohistochemical Analysis of BM Biopsies
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Immunohistochemical staining was performed on BM biopsies. All samples were fixed in formalin mixture, decayed in EDTA, and embedded in paraffin. Serial sections 3μm thick were processed for immunohistochemical staining with antibodies CXCL12, SCF-1, and VEGF-1 (Abcam, USA).
6
Transwell Migration Assay for mMSCs
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The vertical migration of mMSCs was determined using the transwell migration assay. Transwell inserts (6.5 mm diameter and 8 μm pore size; Millipore) were loaded with 1 × 104 mMSCs in 200 μl of serum-free DMEM/F12, and 600 μl of DMEM/F12 supplemented with 10% FBS was added to the lower chambers. In two other groups, 50 ng/ml of CXCL12 (Abcam, Britain) was added to the lower chambers to evaluate the overexpression and suppression of CXCR7 separately. The cells were allowed to migrate at 37 °C in a humidified CO2 incubator for 12 h. The cells remaining on the upper surface of the filter were then removed with cotton swabs, and the cells that migrated to the lower surface were stained with crystal violet (Beyotime Institute of Biotechnology, Haimen, China) for 20 min. Stained cells from five randomly chosen fields were counted under a light microscope.
7
Evaluating Stromal CXCL12 and Vascular Density in Breast Cancer
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TMA slides containing paraffin-embedded breast carcinoma tissues were processed at the Pathology Core Facility and Tissue Archives Human Tissue Resource Network at Ohio State University. A total of 460 samples were included in TMA including 10 adjacent normal or non-malignant lesions. IHC on these slides was performed with CXCL12 (1:100, Abcam), CD31 (1:100, Dako) following standard procedure. Level of CXCL12 expression was scored with Allred score method. Based on Allred scores, 0 and 1 level of CXCL12 expressing samples were grouped as low expression and 1 and 2 level of CXCL12 expressing samples were grouped as high expression samples. CD31+ blood vessels present in a high magnification field were counted manually and samples with ≤ 3 blood vessels were considered as low blood vessel density samples and >3 blood vessels were considered as high blood vessel density samples. Samples were categorized as “Low” when stromal CXCL12 and/or CD31 density was low; samples were categorized as ‘High’ when both stromal CXCL12 and CD31 were high.
8
Western Blot Analysis of Cell Signaling
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Cultured or transfected cells were harvested and washed twice with PBS and lysed in ice-cold radio immunoprecipitation assay buffer (RIPA, Beyotime, Shanghai, China) with freshly added 0.01% protease inhibitor cocktail (Sigma, Shanghai, China) and incubated on ice for 30 min. Cell lysis was centrifuged at 13,000 rcf for 10 min at 4°C and the supernatant (20–30 μg of protein) was run on 10% SDS-PAGE gel and transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). The blots were blocked with 5% skim milk, followed by incubation with antibodies against B7-H4 (Abcam), GAPDH (Fermentas), CXCL12 (Abcam), CXCR4 (Abcam), p-JAK2 and JAK2 (CST), and p-STAT3 and STAT3 (CST). Blots were then incubated with goat anti-mouse or anti-rabbit secondary antibody (Beyotime, Shanghai, China) and visualized using enhanced chemiluminescence (ECL, Thermo Scientific, Shanghai, China).
9
Western Blot Analysis of Signaling Proteins
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For western blot assay, cells were harvested after transfected for 48 hours and lysed with lysis buffer (1% NP-40, 13.7 μg/ml pepstatin A) and mini protease inhibitor at the ratio of 1:7 for 40 min. Then centrifuged at 12 000 rpm for 20 minutes, the concentrations of supernatant proteins were analyzed using the PierceBCA protein assay kit (Thermo scientific, IL, USA). 40 μg of total proteins were conducted electrophoresis in 12% SDS-PAGE gel, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) and incubated with 5% TBST skimmed milk powder. The PVDF membranes were incubated with antibodies against phosphorylated p50 (Abcam, MA, USA) and total p50 (Santa Cruz, CA, USA), CXCL12, CXCR4 or GAPDH (Abcam, MA, USA) for 16 h in 4 °C and washed with TBST 10 min for three times. After that we incubated the PVDF membranes with second antibody for 2 hours in room temperature and washed three times for 10 min each. Finally, immunoblot was visualized using an enhanced chemiluminescence detection system (GE Healthcare life science).
10
Western Blot Protein Analysis
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Cells were collected in the logarithmic growth phase, RIPA lysis buffer was used to extract total cell protein, and the protein concentration was determined by using the BCA kit. 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins and transfer to the PVDF membrane, and they are incubated in 5% skimmed milk for 1 hour. Diluted primary antibodies (α-globulin, γ-globulin, CSF2, CXCR4, DKK1, CXCL12, IGF1, and SCF, 1 : 1000, Abcam, Cambridge, MA, USA) were left overnight at 4°C, then horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 10000, Abcam, Cambridge, MA, USA) was added, and they were incubated for 1 hour. Next, we applied ECL chemiluminescence solution (Beyotime, Shanghai, China) for color development. Images were collected in a gel imaging system.
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