The chondrogenic differentiation procedure was performed as described above, with the addition of the corresponding cytokines. In order to evaluate the chondrogenic differentiation potential upon stimulation with rhIL-1β, rhIL-6 or rhIL-8 (PeproTech), we measured the production of sulfated glycosaminoglycans (sGAG) in the chondrogenic pellets stimulated with rhIL-6 or rhIL-8, and the mRNA expression of SOX9 and MMP13 in the chondrogenic pellets, mediated by rhIL-1β.
Rhil 8
Manufactured by Thermo Fisher Scientific
Sourced in United States
RhIL-8 is a recombinant human interleukin-8 protein. Interleukin-8 is a chemokine that plays a role in immune response and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
24 well transwell plate, by Corning (1 mentions)
Mouse igg1, by Santa Cruz Biotechnology (1 mentions)
Rhe selectin, by R&D Systems (1 mentions)
Anti lfa 1 clone ts2 4, by BioLegend (1 mentions)
Rhp selectin, by R&D Systems (1 mentions)
Rhil 6, by Thermo Fisher Scientific (1 mentions)
Rhil 1β, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Ganetespib, by Selleck Chemicals (1 mentions)
Mts assay kit, by Abcam (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Hexokinase 1, by Cell Signaling Technology (1 mentions)
Lactate dehydrogenase a, by Cell Signaling Technology (1 mentions)
Luminespib, by Selleck Chemicals (1 mentions)
17 aag, by Selleck Chemicals (1 mentions)
Mg132, by Merck Group (1 mentions)
Il 1β elisa kit, by R&D Systems (1 mentions)
Il 8 elisa kit, by R&D Systems (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using rhil 8
1
Chondrogenic Differentiation Modulation
2
Transwell Assay for NB4 Cell Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 24-well transwell plate (Corning, USA) was used to observe the NB4 cell migration induced by Slit2 and IL-8. Based on our experimental results and literature review, NB4 cells were treated with ATRA and fMLP for 48 h. About 0.2 ml of freshly isolated treated NB4 cells (2 × 106 / ml) resuspended in RPMI1640 supplemented with 10% heat-inactivated FBS were transferred to the upper layer of the insert. The upper inserts were placed into the 0.2 ml medium containing NB4 cells treated with 1 uM ATRA and fMLP for 48 h, and then in the RPMI 1640 medium with 0.1% BSA at 37 °C for 2 h. According to previously known results and those from our experiments, the optimum concentration of rh-Slit2 required was 0.4 mg/ml [19] (link) and that of rhIL-8 was 20 pg/ml. rh-Slit2 (150–11, Peprotech, USA) and rhIL-8(200–08 M, Peprotech, USA) were placed in the lower layer and co-cultured in 5% CO2 at 37 °C for 24 h. Cells that migrated through the filter into the lower wells were counted.
3
E-selectin and Integrin Adhesion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rectangular glass capillaries were precoated with 500 µg/ml protein G for 2 h. Capillaries were washed with PBS and coated with 6.6 µg/ml recombinant human E-selectin (rhE-selectin; R&D Systems) and 25 µg/ml mouse IgG1 (Santa Cruz Biotechnology, Inc.), 25 µg/ml activation-specific anti–β2 integrin (clone KIM127; ATCC), or 10 µg/ml anti–LFA-1 (clone TS2/4; BioLegend) for 1 h and subsequently blocked with 1% casein. In another set of experiments, capillaries were coated with 20 µg/ml rhP-selectin (R&D Systems), 50 µg/ml rhIL-8 (PeproTech), and 5 µg/ml mouse IgG1, 5 µg/ml activation-specific anti-β2 integrin (clone mAb24; provided by N. Hogg, Cancer Research UK London Research Institute, London, England, UK), 10 µg/ml anti–LFA-1 (clone TS2/4), 40 µg/ml activation-specific anti–Mac-1 (clone CBRM1/5; BioLegend), or 30 µg/ml anti–Mac-1 (clone M1/70; BioLegend). HL60 cells were resuspended in freshly isolated human plasma. The flow chamber was perfused with the cell suspension for 2 min and subsequently flushed with PBS containing 1 mM MgCl2 and CaCl2 for 1 min to remove nonadherent cells. 10 representative fields of view were recorded, and the number of adherent cells was determined.
4
Investigating HSP90 Inhibitor-Mediated Glycolytic Changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HSP90 inhibitors 17-AAG, luminespib, and ganetespib were obtained from SelleckChem (Houston, TX). CHX and MG132 were purchased from Sigma-Aldrich (St. Louis, MO), and rhIL-8 was purchased from PeproTech (Rocky Hill, NJ). MTS Assay Kit was obtained from Abcam (Cambridge, UK). Glycolysis antibody sampler kit, including PKM2, PFKP, glyceraldehyde-3-phosphate dehydrogenase, hexokinase I (HKI), HKII, lactate dehydrogenase A (LDHA), and PDH antibodies, was purchased from Cell Signaling Technology (Beverly, MA). Antibodies that recognize Ub, PDL1, and IL-8 were purchased from Novus Biologicals (Littleton, CO). Antibodies against IL-1α and IL-1β were ordered from Abcam, and antibodies against HSP90 and β-actin were obtained from Sigma-Aldrich (St. Louis, MO). Antibody that recognizes CD8α was purchased from Cell Signaling Technology. Secondary antibodies were purchased from Invitrogen (Carlsbad, CA). Cell proliferation, viability, colony formation, and Western blot assays were carried out as previously described (18 (link), 43 (link)).
5
Comprehensive Robo1 Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse anti-human Robo1 primary antibody (Mouse IgG1 Clone #770,502, R&D, USA) and PE-conjugated fluorescent-labeled secondary antibody (F0102B, R&D Biotechnology, USA) for measuring the levels of Robo1 using flow cytometry. FITC-conjugated Goat anti-Mouse IgG (H + L) secondary antibody (#GAM001, MultiSciences Biotech Co., Ltd, USA) was used as a reference for Immunofluorescence assay. Mouse monoclonal primary antibody against Robo1 (2 µg/mL, R&D #770,506), human GAPDH(R&D Catalog #AF5718, USA) and anti-mouse IgG secondary antibody (#HAF007, R&D, US) were used for western blotting. PrimeScript™ RT reagent Kit with gDNA Eraser (Cat. RR047A, TaKaRa, Japan) and GoTaq® qPCR Master Mix (#A6002, Promega Corporation, USA) were used to analyze the RNA levels using quantitative real-time PCR. All-trans retinoic acid (ATRA) (R 2625, Sigma Prod., USA) was obtained. Slit2 Quantitative ELISA kit (E-EL-H0931c, Elabscience, China), IL-8 ELISA kit (#Q8000B, R&D, USA), and IL-1β ELISA kit (#DLB50, R&D, USA) were used to measure the levels of Slit2, IL-8, and IL-1β, respectively. rhIL-8 (200–08 M, Peprotech, USA) and rhSlit2-N (150-11, Peprotech, USA) were used to perform the migration assay using transwell.
6
Oral Cancer Cell Line Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cal27, HN4, HN6, HN30, SCC4 cell lines, along with the HIOEC line, were used in this study. All cell lines were recently authenticated by STR profiling and no mycoplasma contamination was detected. SCC4 and Cal27 were purchased from ATCC (Manassas, VA). The cell lines HN4, HN6, and HN30 were kindly provided by the University of Maryland Dental School, USA. All these cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin–streptomycin. rhIL-8 (Peprotech, USA) and recombinant human PTEN (847-PN, R&D, USA) were added to the medium to function as exogenous stimulation. Cells were cultured in a standard humidified atmosphere of 5% CO2 at 37 °C.
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!