Chemotaxis assays across polycarbonate Transwell inserts were performed as previously described (24 (link)). Briefly, 106 CLL PBMCs in AIM-V medium (Gibco) were transferred into the top chambers of Transwell culture inserts (Corning Incorporated) with a diameter of 6.5 mm and a pore size of 5 µm. Filters were then placed into wells containing medium (control), medium with 200 ng/mL CXCL12 and 100 ng/mL CCL19 (Prospec) or a mixture of 50% medium and 50% bone marrow supernatant collected pre-treatment. Where indicated, cells were pre-incubated with 1µM ibrutinib (Sellekchem) in vitro for 1 hour. CLL PBMCs were allowed to migrate for 3 hours at 37°C. Migrated cells in the lower chamber were collected, stained with CD19 and CD3 and counted using AccuCount blank particles (Spherotech) as previously described (25 (link)). The migration index was determined by subtracting the percent migration of the medium control from the percent migration of the chemotractant wells and normalizing. Migration experiments were performed in triplicate plates; results are reported as the average.
Cxcl12
Manufactured by ProSpec
Sourced in Israel
CXCL12 is a chemokine that plays a key role in cell migration and adhesion processes. It is a small secreted protein that binds to the CXCR4 and CXCR7 receptors, regulating various cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
Ibrutinib, by Selleck Chemicals (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Transwell culture insert, by Corning (1 mentions)
Accucount blank particles, by Spherotech (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Thincerttm, by Greiner (1 mentions)
Infinite f500 plate reader, by Tecan (1 mentions)
Metafectene pro, by Biontex (1 mentions)
Antibiotic antimycotic mixture, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse hrp conjugate, by Santa Cruz Biotechnology (1 mentions)
Celastrol, by Alexis Biochemicals (1 mentions)
Trypan blue vital stain, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit horse radish peroxidase conjugate, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
5 protocols using cxcl12
1
CLL Chemotaxis Assay Across Transwell
2
Quantifying CXCR5-Mediated Chemotaxis
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Rhesus macaque PBMCs were transduced with the CAR or CAR/CXCR5 vectors, or mock-transduced. Samples were run in duplicate. For each sample, one million cells in 100 µl X-Vivo-15 media containing 0.1% BSA were placed in the upper chamber of a 24-well plate, with a 5.0-µm transwell membrane (Costar). To the lower chamber containing 600 µl X-Vivo 15 and 0.1% BSA, either CXCL12 at 1 µg/ml or CXCL13 at 2.5 µg/ml (both from ProSpec) were added. No chemokine was added to control wells. After incubation for 4 h at 37°C, cells were collected from the lower chamber, fixed with 1% paraformaldehyde, and counted on a Cytoflex flow cytometer (Beckman). All samples were normalized with the addition of AccuCheck Counting Beads (Invitrogen). Specific cell migration was determined by first subtracting the number of cells that migrated to media alone from the number of cells that migrated to the chemokine and then dividing by the number of cells added to the upper chamber.
3
U266 Cell Transwell Migration Assay
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U266 cells (50 × 104/well) were plated in 300 μl cell culture media with and without 1 μM CSL in the top chambers of 24-well transwell inserts with 8 μm pores (Greiner Bio-One ThinCertTM, Monroe, NC, United States). Cell culture medium (600 μl) containing the Recombinant Human B-cell chemoattractant, CXCL12 (100 ng/ml; ProSpec, Ness-Ziona, Israel) was added to the bottom chamber and incubated for 12 h. Thereafter, the migration assay was performed as described previously (Siveen et al., 2014 (link)).
4
GPCR-Mediated β-Arrestin Translocation Assay
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We used a β-arrestin translocation assay to monitor the movement of a BRET donor-tagged β-arrestin2 from the cytoplasm to the plasma membrane upon GPCR activation as previously described (29 (link)). Briefly, 100,000 HEK293 cells/well in a 96-well opaque microplate (PerkinElmer) were transiently cotransfected with the BRET biosensors Rluc8-β-arrestin2-SP1 (25 ng/well) and M-L-Citrine-SH3 (500 ng/well) with or without 20 ng/well of Flag-CXCR4, Flag-CXCR4-S339fs5, or Flag-CXCR4-R334X using Metafectene Pro (Biontex). Forty-eight hours after transfection, cells were stimulated with increasing concentrations of CXCL12 (ProSpec) in the presence of 5 μM Renilla luciferase coelenterazine H (Cayman Chemical Company) for 20 min. Signals were recorded with a Tecan Infinite F500 plate reader.
5
Celastrol's Anti-cancer Potential Revealed
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Celastrol with purity greater than 98% was purchased from Alexis Biochemicals (San Diego, CA, United States). RPMI 1640, 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture were obtained from Invitrogen (Carlsbad, CA, United States). Propidium iodide (PI), MTT, Tris, glycine, NaCl, SDS, BSA, and β-actin antibody (mouse monoclonal) was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, United States). FBS was purchased from BioWest (Miami, FL, United States). Bortezomib (Velcade, PS341) was purchased from LC Laboratories (Woburn, MA, United States). Nuclear extraction and DNA binding kits was obtained from Active Motif (Carlsbad, CA, United States). Caspase-Glo 3/7 Assay kit was purchased from Promega (Madison, WI, United States). Rabbit polyclonal antibodies against Poly(ADP-ribose) polymerase (PARP), MMP-9, caspase-3, and goat anti-rabbit-horse radish peroxidase (HRP) conjugate and goat anti-mouse HRP conjugate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Rabbit polyclonal antibody against CXCR4 was purchased from Abcam (Cambridge, MA, United States). CXCL12 was purchased from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). ELISA kits for mouse IL-6 and TNF-α were purchased from R&D Systems Inc. (Minneapolis, MN, United States).
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