To test whether CXCR4/CXCL12 regulates in SSC expansion, equal concentration of SSC germ cell culture was plated on STO feeder and treated with the specific CXCR4 inhibitor, AMD3100 (1.25 μM; Sigma), or recombinant CXCL12 (10 ng/mL; R&D Systems), or both. Germ cells were cultured for 7 days after which they were gently dissociated from STO feeders and counted using a hemocytometer. In a repeat experiment, cell viability was assessed using the ATPlite assay kit per manufacturer (Perkin Elmer). In brief, 1 × 104 germ cells were plated onto 96-well plates in defined culture media and treated with AMD3100, or the AKT inhibitor, LY29004 (33 μM). At day 4 and day 6 after treatment, cell viability was assessed by quantifying ATP-dependent luciferase activity.
Recombinant cxcl12
Manufactured by R&D Systems
Sourced in United States
Recombinant CXCL12 is a chemokine protein produced in a laboratory setting. It is involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Amd3100, by Merck Group (2 mentions)
Recombinant il 7, by R&D Systems (2 mentions)
Transwell chamber, by Corning (2 mentions)
Atplite assay kit, by PerkinElmer (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Anti human cd4 magnetic particles, by BD (1 mentions)
2 mercaptoethanol 2 me, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Ifn γ, by BD (1 mentions)
Il 17a, by BD (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Cd93 aa4 1, by BD (1 mentions)
Recombinant cxcl13, by R&D Systems (1 mentions)
Lymphocyte separation medium, by Corning (1 mentions)
Torin1, by Abcam (1 mentions)
Extracellular matrix ecm, by Merck Group (1 mentions)
Rapamycin, by R&D Systems (1 mentions)
12 protocols using recombinant cxcl12
1
CXCR4/CXCL12 Regulation of SSC Expansion
2
T cell Activation and Cytokine Measurement
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Anti-human LRCH1 mAb was purchased from Novus (Littleton, CO, USA). Anti-human CD3, anti-human CD28 was purchased from BD (San Diego, CA, USA). Anti-human CD4 magnetic particles were purchased from BD (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for human TNF-α, IL-17A, IFN-γ, IL-4, and IL-10 were purchased from BioLegend (San Diego, CA, USA). Fluorochrome-conjugated anti-human CD4, IFN-γ, IL-17A, IL-4 and Foxp3 were purchased from BD (San Diego, CA, USA). Recombinant CXCL12 was purchased from R&D Systems (Minneapolis, MN, USA). Phorbol-12-myristate-13-acetate (PMA) and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin, streptomycin, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HERPS), sodium pyruvate and 2-mercaptoethanol (2-ME) were purchased from Life Technologies (Carlsbad, CA, USA).
3
Transwell Migration Assay for T Cell Activation
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PBMC from FAP subjects or healthy donors were activated with TransAct™ (1:100; Miltenyi-Biotec) and recombinant human IL-2 (100 IU/ml; PeproTech) in RPMI-1640 medium supplemented with GlutaMAX-I (Gibco), 5% human serum, 1 mM sodium pyruvate, nonessential amino acids, 10 mM HEPES, 1% penicillin-streptomycin (v/v). At day 5 post stimulation, 3x105 T cells were deposited in the upper inserts of transwell plates (3 µm Pore size, Costar 3415) in RPMI-1640, 0.5% BSA (Nunc). The lower chambers contained the same medium supplemented or not with recombinant CXCL12 (R&D 350-NS; 5 ng/ml). After, 90 min of incubation at 37°C in 5% CO2, inserts were removed, and the same volume of input and migrated cell populations was analyzed by FACS. The percentage of CD4 and CD8 T cells was assessed.
4
Spleen Cell Migration Assay
5
Pancreatic Cancer Cell Line Characterization
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Six human pancreatic cancer cell lines, AsPC-1, BxPC-3, Mia PaCa-2, PANC-1, SU86.86 and T3M4 (kind gifts from Professor Helmut Freiss, Heidelberg University, Germany) were cultured in DMEM or RPMI 1640 media (Hyclone, Thermo Fisher Scientific Inc, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Hyclone), in a humidified incubator with 5% CO2 at 37°C. Extracellular matrix (ECM) and AMD3100, the specific inhibitor of CXCR4 [39 (link)], were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant CXCL12, rapamycin and the antibody against CXCL12 were products of R&D Systems. Torin1 [40 (link)] and antibodies for Western blotting of CXCR7, CXCR4 and VEGF were got from Abcam. The antibody for immunoprecipitation of CXCR7 was produced by Thermo. Other antibodies for immunoblotting were all obtained from Cell Signaling Technology (Beverly, MA).
6
T Cell Adhesion Force Measurement
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Flow chambers (μ-slide, Ibidi) were coated with rhVCAM-1/Fc Chimera (R&D Systems, 862-VC) + recombinant CXCL12 (R&D Systems, 350-NS) (1 μg/ml and 100 ng/ml in PBS, respectively) for 3 hours at RT, rinsed, and equilibrated with RPMI 1640 for 10 min at 37°C and then loaded with T cells for 10 min at 37°C. A flow rate of PBS (0 to 50 ml/min at 37°C) was applied through the temperature-controlled chamber for 92 s using a computer-driven syringe pump (SP210iW, World Precision Instruments) synchronized with image acquisition (three images per second) using an inverted transmission microscope (Axio Observer D1; Zeiss, 10×/0.3-NA objective) and Micro-Manager software (48 ). Images were analyzed using Fiji software (49 (link)) and the Cell Counter plugin. Forces necessary for cells to detach were calculated by determining the flow rate at rupture as described (16 (link)).
7
B Cell Migration Assay Protocol
8
Chemokine-Mediated Cell Migration Assay
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Recombinant CXCL12 (catalogue number 350-NS), CCL19 (catalogue number 361-MI-025), CCL21 (catalogue number 457-6C-025) and anti-human CXCR4 monoclonal antibody (catalogue number MAB173) were purchased from R&D Systems (Abingdon, OX14 3NB, UK). Alexa Fluor 488 anti-human CCR7 antibody (catalogue number 353206) and the recommended isotype control (catalogue number 400233) were purchased from Biolegend (London, NW5 1LB, UK). UltrapureTM low-melting agarose (catalogue number 16520–050) was purchased from Invitrogen (Paisley, PA4 9RF, UK). CXCR4 antagonist AMD310065 (link) (catalogue number 3299), was purchased from Tocris Biosciences (Missouri 63021, USA). CCR7 Antagonist ICT13069 was synthesised at the Institute of Cancer Therapeutics (Bradford, BD7 1DP, UK). Cascade Blue-10 kDa (Catalogue number D1976) and Fluorescein-40 kDa (Catalogue number D1845) dextrans were purchased from ThermoFisher (Loughborough, LE11 5RG, UK). Vectashield hardset antifade mounting medium with DAPI (catalogue number H-1500) was purchased from Vector laboratories, (Peterborough, PE2 6XS, UK). Bovine serum albumin (catalogue number A2153) was purchased from Sigma-Aldrich (Gillingham, SP8 4XT, UK).
9
Splenic B Cell Differentiation Assay
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To mimic TI immunization in vitro, splenic B cells were purified from Mzb1+/+ and Mzb1−/−Prdm1+/gfp mice using anti-B220 magnetic beads (Miltenyi Biotec) and cultured with 25 μg/mL LPS (L5668; Sigma-Aldrich). After 4 d, three populations were isolated: CD138− Blimp− activated B (Act B) cells, CD138− Blimp+ (Pre-PB), and CD138+Blimp+ (PB). In some experiments cells were differentiated in 96-U–bottom-well plates, coated overnight at 4 °C with VCAM-1 (2.5, 5 and 10 µg/mL; R&D Systems). After coating, plates were washed and blocked with Iscove’s modified Dulbecco’s medium (IMDM)-BSA 1% for 1 h at 37 °C. B220+ cells (1 × 105) were added to the wells and cultured with LPS, in the absence or presence of recombinant CXCL12 (0.5 µg/mL; R&D Systems). To differentiate CD138+Blimp+ cells under TD conditions, B220+ cells were cultured for 5 d in the presence of CD40L (5 ng/mL), IL-4, and IL-5 (10 ng/mL; Peprotech).
10
Platelet Aggregation and Signaling Assays
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Ristocetin was purchased from Sigma-Aldrich (Darmstadt, Germany). Recombinant CXCL12, mouse anti-CXCR4 monoclonal antibody, mouse anti-CXCR7 monoclonal antibody, and the soluble CD40 ligand (sCD40L) ELISA kit were purchased from R & D Systems, Inc. (Minneapolis, MN, USA). NSC23766 and Y27632 were purchased from Tocris Bioscience (Bristol, UK) and Calbiochem/Novabiochem Co. (La Jolla, CA, USA), respectively. GAPDH rabbit polyclonal antibody (cat. no. SC-25778) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phospho-specific cofilin antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rac1 and Rho activation assay kits were purchased from Millipore (Billerica, MA, USA). Other materials and chemicals were obtained from commercial sources. Ristocetin was dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the platelet aggregation.
Y27632 and NSC23766 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.3%, which did not affect the platelet aggregation, protein detection by Western blotting or ELISA for sCD40L.
Y27632 and NSC23766 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.3%, which did not affect the platelet aggregation, protein detection by Western blotting or ELISA for sCD40L.
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