Migration and invasion studies were performed using BD Biocoat migration and Matrigel invasion chambers (Becton-Dickson Labware), respectively. Serum free DMEM was added to bottom and top chamber of inserts and allowed to hydrate for 2 h at 37°C with 5% CO2. Next, 0.5 × 105 cells were added to the top chamber of inserts and 100 ng/ml CXCL16 (Peprotech, NJ) was added as chemo-attractant in the bottom chamber. To determine if the migration and invasion of LuCa cells is mediated specifically by CXCR6-CXCL16 interaction, cells pre-incubated with 1.0 μg/ml anti-CXCR6 antibody (MAB699, R&D Systems) were added to the top chamber in one well of Matrigel or control inserts and allowed to migrate/invade under chemotactic gradient of CXCL16 for overnight at 37°C and 5% CO2. After incubation, non-migrating cells on the upper surface of the membrane were removed with a cotton swab. Cells at the bottom surface of the insert were fixed with 100% methanol for 2 min, stained for 2 min with crystal violet (Fisher Scientific), and rinsed twice with de-ionized water. Migrated/invaded cells were counted by microscopy at 40× magnification. All experiments were repeated three times to validate the results.
Cxcl16
Manufactured by R&D Systems
Sourced in United States
CXCL16 is a chemokine that functions as a chemoattractant, promoting the migration of cells expressing the CXCR6 receptor. It plays a role in immune cell trafficking and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Cxcl9, by R&D Systems (2 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions)
Crystal violet, by Thermo Fisher Scientific (1 mentions)
Anti cxcr6, by R&D Systems (1 mentions)
Cxcl16, by Thermo Fisher Scientific (1 mentions)
Mab699, by R&D Systems (1 mentions)
Cxcr6, by R&D Systems (1 mentions)
5.0μm pore polycarbonate membrane, by Corning (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Prolong gold reagent, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Pan cytokeratin, by Bioss Antibodies (1 mentions)
Blocking one reagent, by Nacalai Tesque (1 mentions)
Wnt7a, by R&D Systems (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Pdgf bb, by R&D Systems (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
16 protocols using cxcl16
1
CXCR6-CXCL16 Mediated Cell Migration
2
Monocyte Chemotaxis Assay
3
Immunohistochemical Analysis of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At day 30 after x31 infection, lung tissues were harvested, embedded in OCT compound (Sakura Finetek), and frozen in liquid nitrogen. 6-µm-thick cryosections were prepared and fixed for 10 min with acetone. Fixed sections were rehydrated with PBS, blocked with Blocking One reagent (Nacalai Tesque), and stained with a mixture of Abs against CD45 (30-F11; BD Biosciences), Pan-Cytokeratin (rabbit polyclonal; Bioss), and either CXCL16 (goat polyclonal; R&D Systems) or control Abs (goat IgG; Jackson Immunoresearch) dissolved in PBS containing 2% BSA for 30 min at 37°C. Sections were further incubated with Alexa Fluor–labeled appropriate secondary Abs. After the staining, sections were mounted with Prolong Gold reagent (Life Technologies) and then photographed using an SP5 confocal microscope (Leica Microsystems).
4
Recombinant Growth Factors in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant growth factors were as follows: BMP-7, CXCL16, Dhh, GM-CSF, PDGF-BB, TGFβ1, Wnt3a and Wnt7a (R&D Systems). SB431542 TGFβR1 inhibitor (10 μM) and SB216763 GSK-3β inhibitor (10 μM) are both from Tocris; fibronectin, human plasma are from (341635) Calbiochem and Protein G Dynabeads (10003D) from Life Technologies).
5
Immune Cell Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI-1640 medium containing 10% fetal calf serum was routinely used for primary cell cultures. KnockOut Serum Replacement (Thermo Fisher, Waltham, MA, USA) was used for all migration assays involving S1P. Anti-CD4-PE-Cy7 (GK1.5), anti-CD4-FITC (RM4–5), anti-CD8-PE (53-6.7), anti-CD8-PECF594 (53-6.7), anti-CD69 PerCP-Cy5.5 (H1.2F3), anti-CD44-PE (IM7), anti-CD62L-APC (MEL-14), and anti-p-Stat3-Percp cy5 were purchased from BD PharMingen (San Diego, CA, USA). Anti-CCR2-PE-Cy7 (SA203G11), anti-CD45.2-FITC (104), anti-CD45.1-APC (A20), anti-Qa-2-biotin (695H1-9-9), anti-Qa-2-Alexa Fluor 647 (695H1-9-9), anti-Ki67-PE-Cy7, anti-CXCR3-APC, and anti-CXCR6-PE were purchased from BioLegend (San Diego, CA, USA). Anti-CCR2-APC (Catalog # FAB5538A) and anti-S1P1-PE (Catalog # FAB7089P) were purchased from R&D Systems (Minneapolis, MN, USA). Unlabeled antibodies against Akt, p-Akt (Ser473), STAT3, p-STAT3 (Tyr705), FoxO1 (C29H4), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA). Stat3 inhibitor Stattic was purchased from Selleck.
6
Serum Biomarkers in Murine Lupus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IL-12p40 (BD Biosciences, #555165), CXCL16 (R&D Systems, DY503), IgA (Bethyl laboratories, E90-103), anti-Histone, anti-ANA, and anti-Sm antibodies (Total Ig (A + G + M)) (Alpha Diagnostic International, #5610, #5210, #5405) were measured from mouse serum by ELISA. Serum measurements were performed by 4-point serial dilution (1:2) (as per manufacturer’s protocol) starting at 1:50 of serum from IFNα-accelerated NZB/W or C56BL/6 mice treated with NIK SMI, vehicle, BR3-mIgG2a, or isotype control. Luminex measurements of mouse serum (1:3 dilution) for TNF were done per manufacturer’s protocol (Millipore, #MCYTOMAG-70K-PMX-32).
7
Biomarkers in Metabolic Disorders
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All subjects were assessed after overnight fasting for at least 10 hours. Data on demographic characteristics, medical history, current medications, and blood samples were collected from all subjects at the time of enrollment. Blood samples were immediately centrifuged, separated into aliquots, and stored at −80°C for future batched assays. Serum creatinine, phosphate, and albumin were measured with standard commercial assays. Serum CXCL16 (R&D, Minneapolis, MN, USA) and CRP (Antibody and Immunoassay Services, HK)concentrations were measured in duplicate with commercially available enzyme-linked immunosorbent assays according to the manufacturers' instructions in the Core Laboratory of School of Pharmacy, Wenzhou Medical College. All other clinical biochemistry tests were processed in the Clinical Examination Laboratory of the 2nd Affiliated Hospital of Wenzhou Medial College after a single thaw.
8
Monocyte Isolation and Transfection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For proof-of-concept experiments, monocytes were isolated from the blood of healthy donors using StraightFrom Whole Blood CD14 MicroBeads and Whole Blood Column Kit as described above. Cells were seeded in 24-mm diameter culture plates at a density of 7 × 105 cells/well in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% FBS (Lonza, Verviers, Belgium) and 1% Pen-Strep (Lonza).
For transfection experiments, cells seeded in 750 μl of RPMI 1640 (GIBCO) were transfected with Lipofectamine RNAiMAX Reagent using either Pre-miR miRNA Precursor Negative Control as a scrambled control or Pre-miR-hsa-miR-451a Precursor (30 pmol) (all Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions and incubated at 37 °C with an atmosphere containing 5% CO2 for 24 h. For stimulation experiments, the cells were stimulated with 5 or 50 ng of CXCL16 (R&D Systems, Inc., Minneapolis, MN, USA) or the corresponding control and incubated at 37 °C with an atmosphere containing 5% CO2 for 6 and 24 h. Cell culture supernatants were separated after centrifugation for 10 min and stored at − 80 °C until analysis.
For transfection experiments, cells seeded in 750 μl of RPMI 1640 (GIBCO) were transfected with Lipofectamine RNAiMAX Reagent using either Pre-miR miRNA Precursor Negative Control as a scrambled control or Pre-miR-hsa-miR-451a Precursor (30 pmol) (all Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions and incubated at 37 °C with an atmosphere containing 5% CO2 for 24 h. For stimulation experiments, the cells were stimulated with 5 or 50 ng of CXCL16 (R&D Systems, Inc., Minneapolis, MN, USA) or the corresponding control and incubated at 37 °C with an atmosphere containing 5% CO2 for 6 and 24 h. Cell culture supernatants were separated after centrifugation for 10 min and stored at − 80 °C until analysis.
9
Cytokine-Induced Id1 Expression in Synovial Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HMVECs, EPCs, monocytes, and RA, OA, and NL synovial fibroblasts were plated in 6-well plates at 200,000 cells/well and serum starved overnight. Media was exchanged and collected after 24 h. The collected media was analyzed by ELISA for Id1 (MyBioSource, San Diego, CA, USA). Manufacturer protocols were followed. SFs of RA, OA, and several other diseases were also analyzed by this ELISA. Both synovial and dermal fibroblasts were stimulated with varying cytokines and concentrations. Cytokines used were tumor necrosis factor alpha (TNF-α, Thermo Fisher Scientific), chemokine (C-X-C motif) ligand 16 (CXCL16, R&D Systems, Minneapolis, MN, USA), interleukin 17 (IL-17, R&D Systems), and TGF-β (R&D Systems). These cytokines were chosen because they are known to be upregulated in RA ST [23 (link), 24 (link)]. The supernatant and exosome fractions were collected after 24 h and analyzed by this ELISA.
10
ELISA Quantification of Angiogenic Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!