CLL PBMCs were seeded at 5 × 105 cells/200 μL of complete media (Media199, 10% fetal calf serum (FCS), penicillin/streptomycin and 5 ng/mL interleukin-4 [RayBiotech, Peachtree Corners, GA, USA]). Cells were cultured ±1 μM ODN2006 (TLR9 agonist; InvivoGen, San Diego, CA, USA) and incubated for 24 h. For FAK inhibition, CLL cells were pre-incubated for 2 h with Defactinib (Selleck Chemicals, Houston, TX, USA) at a range of concentrations (0.5 µM–5 µM). After 24 h, the amount of apoptosis was determined using a FITC-labeled Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend). The p-FAK levels in the CLL cells were measured by intracellular staining with a PE-conjugated anti-p-FAK antibody (BD Biosciences, Franklin Lakes, NJ, USA), after fixing and permeabilizing the cells using the True-Phos™ kit (BioLegend) according to the manufacturer’s instructions. The MFI values for p-FAK were determined in gated CD19+/CD5+ CLL cells using a Cytoflex LX flow cytometer (Beckman Coulter).
Interleukin 4 (il 4)
Manufactured by RayBiotech
Sourced in United States
IL-4 is a protein that plays a central role in the regulation of immune and inflammatory responses. It is a cytokine produced by a variety of cell types, including T cells, mast cells, and eosinophils. IL-4 is involved in the differentiation of naive helper T cells into Th2 cells and the stimulation of B cell growth and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Il 10, by RayBiotech (4 mentions)
Il 13, by RayBiotech (4 mentions)
Interleukin 6 (il 6), by RayBiotech (3 mentions)
Defactinib, by Selleck Chemicals (2 mentions)
Odn2006, by InvivoGen (2 mentions)
Media199, by RayBiotech (2 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (2 mentions)
Mcp 1, by RayBiotech (2 mentions)
Tnf α, by RayBiotech (2 mentions)
Il 1β, by RayBiotech (2 mentions)
Interleukin 2 (il 2), by RayBiotech (2 mentions)
Cxcl12, by BioLegend (1 mentions)
24 well plate, by Corning (1 mentions)
20 r rg3, by Yuanye Bio-Technology (1 mentions)
Cell cycle kit, by BD (1 mentions)
Cyclophosphamide cp, by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Automated cell counter, by Beckman Coulter (1 mentions)
Vegf a, by RayBiotech (1 mentions)
Tgf β, by MyBioSource (1 mentions)
8 protocols using interleukin 4 (il 4)
1
Measuring FAK Inhibition in CLL Cells
2
CLL Cell Migration Assay with TLR9 Agonist and Defactinib
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CLL PBMCs were seeded at 5 × 105 cells/200 μL of complete media (Media199, 10% fetal calf serum (FCS), penicillin/streptomycin and 5 ng/mL interleukin-4 [RayBiotech]). Cells were cultured ±1 μM ODN2006 (TLR9 agonist; InvivoGen) ±1 μM defactinib (Selleck Chemicals) and incubated overnight. Transwell migration assays were performed using polycarbonate transwell inserts (5 μm pores) in 24-well plates (Corning Inc., Corning, NY, USA). A total of 600 μL complete media + 100 ng/mL CXCL12 (BioLegend) were added to the basolateral chambers, and PBMC (200 μL complete media) from CLL patients were transferred into the apical chambers and incubated for 4 h. Migrated CD19+/CD5+ CLL cells were quantified by volumetric counting using a Cytoflex LX flow cytometer (Beckman Coulter).
3
Quantification of Ginsenosides and Monosaccharides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reference substances of ginsenosides (nR1, Rg1, Re, pF11, Rf, Ra2, Rb1, Rc, Ro, Rb2, Rb3, Rd and 20(R)-Rg3) and monosaccharides (Glc, Ara, Gal, Man, GalA, Rha, Rib, ClcUA, Xyl and Fuc) were purchased from Shanghai Yuanye Biotechnology (Shanghai, China). The purity of these references was higher than 99.0% indicated by HPLC analysis. Cyclophosphamide (CP) was supplied by Sigma-Aldrich Company Ltd., (United States). Thrombopoietin (TPO), erythropoietin (EPO), and granulocyte-macrophage colony stimulating factor (GM-CSF) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Quantibody®Array Glass Chip of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IFNγ, TNF-α and MCP-1 were purchased from RayBiotech Life, Inc. (United States). FITC-Annexin-V and Cell Cycle kits were purchased from BD Biosciences Pharmingen (United States). The blood cell analysis reagent kit was purchased from IDEXX Laboratories Inc (United States). Protopanaxadiol saponins (PDS) and protopanaxatriol saponins (PTS) were supplied by Professor Chen Yanping in the College of Chemistry, Jilin University; PDS and PTS were LC-MS grade. The voucher specimen of PDS (S20190011) and PTS (S20190012) were deposited in the authors’ lab in Changchun University of Chinese Medicine (Changchun, China).
4
Plasma Biomarker Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasma samples were obtained by venipuncture after an overnight fasting. Glucose was analyzed by an automated analyzer (Hitachi Modular P800, Roche, Basel, Switzerland). Serum concentrations of triglycerides and free fatty acids (FFA) were determined by using commercially available kits (Infinity, Thermo Electron Corporation, Melbourne, Australia). The carcinoembryonic antigen (CEA), fibrinogen and high sensitivity C-reactive protein (CRP) concentrations were measured as previously reported [11 (link)]. White blood cell (WBC) count was determined using an automated cell counter (Beckman Coulter, Inc., Fullerton, CA, USA). Circulating levels of MAGP-1, IL-4, IL-6, IL-13, VEGFA, OPN (RayBiotech, Inc., Norcross, GA, USA) and TGF-β (Mybiosource, San Diego, CA, USA) were assessed by commercially available ELISA kits according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were <10 and <12% for all analysed molecules.
5
Cytokine Profiling in Serum Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recipient serum was obtained from peripheral blood 10 days before surgery and when euthanasia was performed or death observed. Cytokine concentrations were measured by ELISA kits as described by the manufacturers, including comparisons with standard curves. Kits used and minimum detectable concentrations of interleukins (IL): IL-4: 1.5 pg/ml (RayBiotech Inc., Norcross, GA, USA); IL-10: 10 pg/ml (RayBiotech Inc., Norcross, GA, USA); IL-12: 9.375 (Elabscience, Bethesda, MD, USA); IL-17: 23.43 pg/ml (Cusabio, Hubei, China); IL-21: 3.3 pg/ml (Merck-Millipore, Billerica, MA, USA); IL-23: 0.196 pg/ml (Merck-Millipore, Billerica, MA, USA); tumor necrosis factor-alpha (TNF-a): 15 pm/ml (Diaclone, Besancon Cedex, France); and transforming growth factor (TGF)-b1: 11.4 pg/ml (Milliplex ® , Merck-Millipore, Billerica, MA, USA).
6
Multiplex Analysis of Rat Synovial Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were euthanized via CO2 inhalation at three days or twenty-one days post-surgery for both the naïve group and the MMT surgery group. Synovial fluid was collected by first injecting 100 μl of saline intra-articularly using a 30 gauge insulin syringe, followed by aspirating approximately 50 to 100 μl of the synovial fluid and saline using the same syringe. Synovial fluid was analyzed using the Quantibody® Rat Inflammation Array 1, a multiplex ELISA kit that quantitatively measured 10 rat inflammatory factors: IFNγ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, monocyte chemoattractant protein-1 (MCP-1), and TNFα (RayBiotech, Norcross, GA, USA).
7
Paracrine Effect of ADRCs on Macrophage Polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the paracrine effect of ADRCs on macrophage polarization, a trans-well co-culture system (Cell Culture Insert plates; Thermo Fisher Scientific, Tokyo, Japan) was used. In this system, macrophages were seeded in the base of 12-well plates and ADRCs were seeded on the insert. Thus, only factors secreted from the cells would pass the membrane. To induce M1 polarization, 50 ng/mL IFN-γ (RayBiotech, Norcross, GA, USA) and 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) were added to the basal medium, and 20 ng/mL IL-4 (RayBiotech) was added to induce M2 polarization under the following conditions.
First, macrophages were seeded at a density of 1 × 105 cells/well in the aforementioned 12-well plates and cultured for two days. Macrophages were then divided into five groups and cultured for two days under the following conditions: control group, basal medium only; M1-polarized group, IFN-γ/LPS stimulation; M1-polarized + ADRC group–IFN-γ/LPS stimulation + co-culture with ADRCs (2 × 104 cells/well); ADRC group, co-culture with ADRCs (2 × 104 cells/well); M2-polarized group, IL-4 stimulation. A summary of the grouping is presented inFigure 1 .
First, macrophages were seeded at a density of 1 × 105 cells/well in the aforementioned 12-well plates and cultured for two days. Macrophages were then divided into five groups and cultured for two days under the following conditions: control group, basal medium only; M1-polarized group, IFN-γ/LPS stimulation; M1-polarized + ADRC group–IFN-γ/LPS stimulation + co-culture with ADRCs (2 × 104 cells/well); ADRC group, co-culture with ADRCs (2 × 104 cells/well); M2-polarized group, IL-4 stimulation. A summary of the grouping is presented in
8
Isolation and Polarization of Rat Bone Marrow-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The femur was isolated from euthanized Wistar male rats and marrow was flushed into a cold PBS solution containing 2% fetal bovine serum (FBS). Mechanically dissociated cells were incubated on ice with three volume of 0.8% NH4Cl solution to remove red blood cells. 106 cells/cm2 were seeded in tissue culture plates (Corning Constar) and cultured in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) supplemented with 10% FBS, 1% v/v penicillin-streptomycin (Life Technologies) 10ng/ml M-CSF (RayBiotech). After 7 days, bone marrow-derived macrophages (MΦ) were polarized to inflammatory M1 with IMDM, 1% v/v penicillin-streptomycin, 10ng/ml M-CSF supplemented with 100ng/ml TNFα (ApexBio) and 100ng/ml IFNγ (RayBiotech). The non-inflammatory BMDM-M2 profile was induced by supplementing the culture medium with 20ng/ml IL-4, 20ng/ml IL-10 and 20ng/ml IL13 (RayBiotech).
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!