Sdf1a

Manufactured by R&D Systems

Sourced in Germany

SDF1a is a recombinant human chemokine protein. It functions as a chemoattractant for a variety of cell types, including lymphocytes, monocytes, and stem cells.

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Lab products found in correlation

9 protocols using sdf1a

Labeling and Binding Assay of Murine Proteins

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Purified murine IgG (RayBiotech), murine recombinant TFF2 (Peprotech) and SDF1a (R&D systems) were labeled with Alexa Fluor 647 dye using the Alexa Fluor 647 protein labeling kit (A30009, Molecular Probes) per manufacturer’s directions. HEK293 cells were maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) at 37°C with 5% CO2. HEK293 cells were seeded at 1 × 10⁶/well and incubated at 37°C with 5% CO2 overnight before transfection with 3–6ug of plasmids pCMV3-mLINGO3-GFP, pCMV3-mLINGO2-GFP or pCMV3-mCXCR4-GFP using FuGENE HD DNA transfection reagent (Promega) based on manufacturer’s instructions. 24h post transfection, cells were incubated with either PBS or 3ug of Alexa Fluor 647 labeled soluble ligands for 16h at 4°C. Cells expressing GFP and Alexa Fluor 647 positive were identified by flow cytometry.

Zullo K.M., Douglas B., Maloney N.M., Ji Y., Wei Y., Herbine K., Cohen R., Pastore C., Cramer Z., Wang X., Wei W., Somsouk M., Hung L.Y., Lengner C., Kohanski M.H., Cohen N.A, & Herbert D.R. (2021). LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis. Scandinavian journal of gastroenterology, 56(7), 791-805.

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Quantification of Angiogenic Factors

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Colorimetric ELISA kits were used for SDF-1a (R&D Systems, Minneapolis, MN) and multi-analyte electrochemoluminescence ELISA kits were used for VEGF-A, C, & D, sFlt-1, and bFGF (MesoScale Discovery, Rockville, MD). All assays were run according to the manufacturer instructions. All plasma samples were assayed in duplicate and averaged. The lower limits of quantification (in pg/mL) were 47 for SDF-1a, 5 for VEGF-A, 146 for VEGF-C, 67 for VEGF-D, 10 for sFlt-1, and 3 for bFGF.

Sandeep N., Uchida Y., Ratnayaka K., McCarter R., Hanumanthaiah S., Bangoura A., Zhao Z., Oliver-Danna J., Leatherbury L., Kanter J, & Mukouyama Y.S. (2015). CHARACTERIZING THE ANGIOGENIC ACTIVITY OF SINGLE VENTRICLE PATIENTS WITH AORTOPULMONARY COLLATERAL VESSELS. The Journal of thoracic and cardiovascular surgery, 151(4), 1126-1135.e2.

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Neural Stem Cell Migration Assay

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Neurospheres in the range of 100–200 μm were seeded on coverslips coated with as before to evaluate the influence of coating concentration on expansion of the neurosphere as a measurement of migration. Alternatively, fixed concentrations of 50 μg/ml of either PDL or PornT was used to evaluate the response of the spheres to proteins known to influence the migration and differentiation of NPCs depending on the coating of choice. In the latter case neurospheres were stimulated with 500 ng/μl of stromal-cell derived factor-1alpha (SDF-1a, R&D systems, Germany) or 50 IU/ml of rhEpo (NeoRecormon, Roche, Switzerland). Expansion was tracked for 8 h on a Zeiss inverted microscope equipped with a cell culture chamber. Images were taken every 10 min with a 10x phase contrast objective. Expansion of the spheres was analyzed using custom designed macros on ImageJ. The increase in area occupied by the neurosphere was calculated as

r A = \frac{f A}{i A}

, where, rA is the ratio of the sphere area, fA is the area at the end of the recording, and iA is the area at the beginning of the recording. Data is represented as ratio of the respective control (Figures 7 and 8) or as rA (Supplementary Figure S1). At least 10 neurospheres were tracked in a total of 3–4 experiments per evaluated condition.

Sanchez-Mendoza E.H., Schlechter J., Hermann D.M, & Doeppner T.R. (2016). Characterization of Seeding Conditions for Studies on Differentiation Patterns of Subventricular Zone Derived Neurospheres. Frontiers in Cellular Neuroscience, 10, 55.

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Ligand Binding and Receptor Expression

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Purified murine IgG (RayBiotech), murine IL-2, murine recombinant TFF3 (Peprotech) and SDF1a (R&D systems) were labeled with Alexa Fluor 647 dye using the Alexa Fluor 647 protein labeling kit (A30009, Molecular Probes) per manufacturer’s directions. HEK293 cells were maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) at 37 °C with 5% CO₂. HEK293 cells were seeded at 1 × 10⁶/well and incubated at 37 °C with 5% CO₂ overnight before transfection with 3ug of plasmids pCMV3-mTLR2-GFP, pCMV3-mLINGO2-GFP or 6ug of pCMV3-mCXCR4-GFP using FuGENE HD DNA transfection reagent (Promega) based on manufacturer’s instructions. 24 h post transfection, cells were incubated with either PBS or 3 μg of Alexa Fluor 647 labeled soluble ligands for 16 h at 4 °C. Cells expressing GFP and/or Alexa Fluor 647 positive were identified by flow cytometry using a LSRII.

Belle N.M., Ji Y., Herbine K., Wei Y., Park J., Zullo K., Hung L.Y., Srivatsa S., Young T., Oniskey T., Pastore C., Nieves W., Somsouk M, & Herbert D.R. (2019). TFF3 interacts with LINGO2 to regulate EGFR activation for protection against colitis and gastrointestinal helminths. Nature Communications, 10, 4408.

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Optimizing Cell Migration Assays

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Migration assays were performed in a migration chamber (Millipore, Burlington, MA) according to the manufacturer’s directions using epidermal KCs, Swiss mouse 3T3 cells (ATCC, Manassas, VA), HMGB1 (BioLegend, San Diego, CA; #764004), or SDF1a (R&D Systems, Minneapolis, MN; Cat# 460-SD/CF) as bait. Positive controls were epidermal KCs, and negative controls were included medium without serum or growth factors. In addition, to confirm the specificity of HMGB1 as bait, HMGB1 antibody (Invitrogen, Carlsbad, CA; #PA5-29604) was added in the presence of HMGB1 (10 and 50 ng/ml), and non-antibody-treated HMGB1 group served as a positive control. Preliminary experiments were performed to establish optimum dosage and migration times, and experiments were conducted with triplicate wells and were repeated three times.

Park H., Lad S., Boland K., Johnson K., Readio N., Jin G., Asfaha S., Patterson K.S., Singh A., Yang X., Londono D., Singh A., Trempus C., Gordon D., Wang T.C, & Morris R.J. (2018). Bone marrow-derived epithelial cells and hair follicle stem cells contribute to development of chronic cutaneous neoplasms. Nature Communications, 9, 5293.

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Transwell-based Neutrophil Migration Assay

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Cell migration assays were performed in 24-well Transwell chambers (Corning Costar, Cambridge, MA) using polycarbonate membranes with a pore size of 5 μm. Approximately 200,000 neutrophils were labeled in 1 mL of PBS containing 10 µM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA, USA) for 5 min at room temperature and were loaded into the upper chambers. Medium or medium supplemented with 10% FBS, HCC-CAFs CM, SDF1a (50 ng/mL, R&D Systems, Minneapolis, MN), SDF1a blocking antibody(10 μg/mL, R&D Systems, Minneapolis, MN), and CXCR4 blocking antibody was added to the lower chambers. After the cells were incubated for 6 h at 37 °C with 5% CO₂, the number of cells in the lower chamber was determined.

Cheng Y., Li H., Deng Y., Tai Y., Zeng K., Zhang Y., Liu W., Zhang Q, & Yang Y. (2018). Cancer-associated fibroblasts induce PDL1+ neutrophils through the IL6-STAT3 pathway that foster immune suppression in hepatocellular carcinoma. Cell Death & Disease, 9(4), 422.

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Protein Expression Analysis in Diaphragm

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Samples designated for ELISA test (5 for each group) were snap frozen and stored at −80 °C until the beginning of the assay. Before start, tissue was thawed, lysed by mechanical homogenization, and immediately assayed. Nonoperated B6 diaphragms served as control. Three decellularized diaphragm quarters (from different samples) were pooled and used for each ELISA. Quantikine for mouse VEGF, HGF, EGF, and SDF-1a (R&D systems) were carried out according to manufacturers’ instructions. Luminescence acquisition and sample quantification was performed using SpectraMax Plus 384 (Molecular devices, San Jose, CA, USA).

Alvarèz Fallas M.E., Piccoli M., Franzin C., Sgrò A., Dedja A., Urbani L., Bertin E., Trevisan C., Gamba P., Burns A.J., De Coppi P, & Pozzobon M. (2018). Decellularized Diaphragmatic Muscle Drives a Constructive Angiogenic Response In Vivo. International Journal of Molecular Sciences, 19(5), 1319.

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Transwell Migration and Cell Adhesion Assays

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For Transwell migration assays, stromal cell-derived factor 1 (SDF1a; R&D Systems, Minneapolis, Minn) and CCL21 (R&D Systems) were added to the lower chamber, and 200,000 cells were placed in the upper chamber. Cells were collected in the lower chamber after 4 hours at 378C. Migrated cells were counted (by using AccuCheck Counting Beads, Life Technologies) and analyzed by means of flow cytometry. All experiments were performed in triplicate. For the cell adhesion assays, wells were coated with retronectin-CH 296 (Takara, Shiga, Japan) or vascular cell adhesion molecule 1 (R&D Systems), and 200,000 cells were incubated for 1 hour at 378C. Adherent cells were collected (after washing with PBS with 2 mmol/L EDTA) and counted. For the detection of the high-affinity lymphocyte function-associated antigen 1 (LFA-1), cells were cultured in the presence of 10 mmol/L EDTA (the nonactivated condition) or 1 mmol/L MgCl 2 (the activated condition) plus SDF1a, as previously described. 26 After 5 minutes of activation, cells were stained with the A579 ligand mimetic antibody (kindly provided by T. A. Springer, Harvard Medical School, Boston, Mass) and analyzed by means of flow cytometry.

Lagresle-Peyrou C., Luce S., Ouchani F., Soheili T.S., Sadek H., Chouteau M., Durand A., Pic I., Majewski J., Brouzes C., Lambert N., Bohineust A., Verhoeyen E., Cosset F.L., Magerus-Chatinet A., Rieux-Laucat F., Gandemer V., Monnier D., Heijmans C., van Gijn M., Dalm V.A., Mahlaoui N., Stephan J.L., Picard C., Durandy A., Kracker S., Hivroz C., Jabado N., de Saint Basile G., Fischer A., Cavazzana M, & André-Schmutz I. (2016). X-linked primary immunodeficiency associated with hemizygous mutations in the moesin (MSN) gene. The Journal of allergy and clinical immunology, 138(6).

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Evaluating Endothelial Progenitor Cell Migration

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Migration activity of cultured EPCs was evaluated with a chemotaxis chamber assay as described previously (12). Briefly, the polycarbonate filter (5-mm pore size) (Transwell; Corning, Corning, NY, USA) was placed between the upper and lower chambers. The cultured EPCs were coincubated with recombinant mouse sFlt1 protein (300 ng/ml; R&D Systems, Minneapolis, MN, USA) for 24 h in 0.5% FBS/endothelial basal medium 2 (EBM2) (Lonza, Tokyo, Japan) before migration assay. The sFlt1-pretreated EPC suspensions ( 5´ 10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with 0.5% FBS/EBM2 (Lonza) with growth factors alone or that with a chemoattractant SDF-1a (100 ng/ml) (R&D Systems). The cells were incubated for 16 h at 37°C in 5% CO 2 . The total number of migrated cells on the lower chamber side of polycarbonate filter was evaluated in each well and expressed as the migration activity. The polycarbonate filter with migrated cells was fixed with 2% PFA/PBS and stained with 4,6-diamidinophenylindole (DAPI) (Sigma-Aldrich) to visualize nuclei. The DAPI-positive nuclei were counted in three polycarbonate filters and averaged for comparison. The experiment was repeated for over three times, and the representative result was demonstrated.

Kanki K., Ii M., Terai Y., Ohmichi M, & Asahi M. (2016). Bone Marrow-Derived Endothelial Progenitor Cells Reduce Recurrent Miscarriage in Gestation. Cell transplantation, 25(12).

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