After LEM characterization, mouse recombinant SDF‐1α (PeproTech) was loaded into the scaffold according to Namiri et al. (2018[32 (link)]) using an m‐maleimidobenzoyl‐N‐hydroxysulfosuccinimide ester (sulfo-MBS). For this purpose, the LEM slices were immersed in 1 mg/ml sulfo‐MBS in PBS for 1 h, then incubated in a solution of 500 ng/ml SDF‐1α in PBS for 4 h. Free SDF‐1α was removed by washing three times with PBS. Finally, the amount of immobilized SDF‐1α was measured by quantification of remaining growth factor in the washing solutions by using an SDF‐1α sandwich enzyme‐linked immunosorbent assay (ELISA, MCX120 R&D Systems) kit.
Sdf 1α
Manufactured by R&D Systems
Sourced in United States, United Kingdom
SDF-1α is a recombinant human chemokine. It is a small cytokine that attracts and activates various cell types, including leukocytes, hematopoietic stem cells, and endothelial cells. SDF-1α is involved in the regulation of cell migration and homing.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by R&D Systems (4 mentions)
Protein a g, by Thermo Fisher Scientific (4 mentions)
Ccl20, by R&D Systems (3 mentions)
Rantes, by R&D Systems (3 mentions)
Mcp 1, by R&D Systems (3 mentions)
Amd3100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Transwell chamber, by Corning (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Spotchem automatic dry chemistry system, by Arkray (2 mentions)
Transforming growth factor β1 tgf β1, by Abcam (2 mentions)
Inf γ, by Abcam (2 mentions)
Interleukin 2 (il 2), by Abcam (2 mentions)
C reactive protein crp, by Abcam (2 mentions)
High mobility group box 1 hmgb1, by LifeSpan BioSciences (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Mip 1α, by R&D Systems (2 mentions)
63 protocols using sdf 1α
1
SDF-1α Immobilization on LEM Scaffold
2
SDF-1α Gradient Generation for Cell Attraction
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Two hours after MSC seeding, cell culture medium containing 250ng/ml of SDF-1α (R&D Systems, MN, USA) was applied only to the conditioning channel to generate an SDF-1α gradient within the collagen gel matrix. The concentration of SDF-1α in vivo is known to be about 0.5 to 0.8 ng/ml in human circulation and 1 to 5 μg/ml in in vivo fluids. However, the optimal concentration range for most chemokines for in vivo cell attraction is 10 to 1000 ng/ml [19 (link)]. Here, 250ng/ml of SDF-1α was applied every 24 hours after washing the channels with media in order to reset the gradient.
3
NT21MP Enhances Cell Invasion In Vitro
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The cells were seeded in six-well plates until the cells grew to 90% confluence. Wounds were created by a 10-µl pipette tip and washed with PBS. Then, some of the cells were treated with 1 µg/ml NT21MP for 24 h. Images were taken at 0 and 24 h. The invasive potential of cells was analyzed using Transwell chambers. Briefly, cells were added to the upper chamber, then cell culture medium with 10% FBS was added to the lower chamber and cultured for 20 h at 37°C. The cells on the underside were fixed with 4% paraformaldehyde and stained with Giemsa solution. The number of stained invasive cells was photographed under a microscope at 400× magnification (Olympus IX71, Tokyo, Japan). In addition, the methods being added between NT21MP and SDF-1α were as following: the cells were treated with 1 µg/ml NT21MP for 30 min at 4°C and 30 min at 37°C, then added SDF-1α (100 ng/ml, R&D Systems, Minneapolis, MN) at room temperature.
4
Chemotactic Potency of Recombinant SDF-1α
5
Bioactive Hydrogel Degradation and Release
6
Chemotaxis Assay for DLBCL Cells
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DLBCL cell lines were treated with vehicle, R406 (1 μM), GDC-0941 (0.5 μM), Ibrutinib (0.1 μM), AMD3100 (10 μM) or R406 + AMD3100 for 24 h. Before the chemotaxis assay, Permeable Polycarbonate Membrane Inserts in the Corning™ Transwell™ 24-well plate (Fisher Scientific, pore size 8 mm) were pretreated by adding 600 μL of RPMI-1640 media containing 0.5% bovine serum albumin (Sigma) with or without SDF-1α (25-100 ng/mL, R&D Systems) into the bottom chamber at 37°C for 1 h. Each lot of SDF-1α was individually titrated for activity in the chemotaxis assay prior to use. Treated cells were harvested and resuspended in RPMI-1640 media for a final density of 2×106/mL. 100 μL of cell suspension was transferred to each top chamber and incubated at 37 °C for 2-4 h. Cells in the lower chambers were harvested and cell numbers were determined by manual counting. Each condition was set up in triplicate.
7
Evaluating Paclitaxel-Resistant Breast Cancer Cell Invasion
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The invasive activity of paclitaxel-resistant breast cancer cells treated with NT21MP or PDGFR inhibitor was evaluated using Transwell inserts as described previously [26 (link)]. 600 μL serum-free medium with SDF-1α (100 ng/mL, R&D Systems, Minneapolis, MN) was added to the bottom wells of the 24-well plate, and no SDF-1α was used in the blank group. One million SKBR-3 cells resuspended in buffer with NT21MP or PDGFR inhibitor were added to the upper chamber insert. After 24 h, the cells in the upper chamber had partly invaded the underside. The invaded cells were fixed with 4% paraformaldehyde, stained with Giemsa solution, and photographed under a microscope.
8
Imaging Bone Tissue Structure and Vascularization
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Tibias and femurs were fixed in 4% PFA with 10% EDTA for 4–8 h on ice in 4°C while rotating, followed by overnight incubation in 20% EDTA in 4°C. Next, the bones were put to sucrose solutions in PBS: 10%—2 h, 20%—2 h, and 30%—overnight in 4°C. Next, the excess of sucrose was removed, and bones were embedded in Tissue‐Tek Oct media (VWR) and frozen in dry‐ice‐cooled isopentane (Sigma). 70‐μm‐thick longitudinal section of bones were cut on cryostat (Leica), blocked with 5% goat or donkey serum and 3% BSA, and stained overnight with primary antibodies in 4°C, followed by 1‐h staining with secondary antibodies in room temperature. Primary antibodies used for immunohistochemistry include HO‐1 (cat. SPA894, Enzo Life Sciences), CD31 (clone MEC13.3, BD Biosciences), endomucin (clone V.7C7), Sca‐1 (cat. AF1226, R&D), SDF‐1α (cat. sc‐6193, Santa Cruz Biotechnology), and CD140β (clone APB5, eBioscience). Secondary antibodies used in the study include donkey F(ab’)2 fragments (Jackson ImmunoResearch) and donkey or goat whole IgG fragments (Molecular Probes) conjugated with Alexa Fluor dyes or Cy3 dyes. Imaging was done on LSM780 confocal microscope (Zeiss) and analyzed in ImageJ software.
9
Transwell Migration and Invasion Assays
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Transwell migration assays were performed using 24-well transwell inserts (8 μm pore size; Costar, USA). Cells (5 × 104) were seeded in the upper inserts, which were placed in 24-well plates containing EGM-2 with 100 ng/ml SDF-1α (R&D Systems, USA) and incubated for 6 h at 37°C. Cell migration activity was quantified by counting the number of cells that migrated in five random microscopic fields (200× magnification). For invasion assays, 5 × 104 cells were placed in the upper inserts coated with Matrigel (BioCoat Matrigel Invasion Chamber; USA). The cells were incubated in EGM-2 containing stromal cell-derived factor 1-alpha (SDF-1α) for 24 h at 37°C, and then the number of invaded cells was quantified in the same way described above for the transwell migration assay.
10
Exploratory Plasma Biomarkers of Cabozantinib
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