Human lung microvascular endothelial cells (HMVEC-l, Lonza, Basel, Switzerland) in the fourth passage were grown to 80% confluence in T75 flasks (Greiner Bio-One, Kremsmünster, Austria) containing 10 mL endothelial basal medium and growth supplements (Lonza). Where required, recombinant TNFα (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 50 ng/mL and incubated for 10 h at 37 °C and 5% pCO2. TNFα incubation times and dosage have been optimized recently in our labs [69 ]. Where required, heparinase III (0.1 mU/mL, Iduron, Alderley, UK) and chondroitinase ABC (0.5 mU/mL, Sigma-Aldrich) were added to the culture medium after 30 min of incubation with TNFα. To rule out CXCL-8 signaling through CXCR1 and CXCR2 and binding to DARC/D6, 0.5 μg/mL of each anti-CXCR1, anti-CXCR2 and anti-DARC/D6 antibody (Santa Cruz, Dallas, TX, USA) were added to the medium. After incubation for 90 min, recombinant CXCL-8 (Antagonis Biotherapeutics GesmbH, Graz, Austria) was added to the medium at a final concentration of 50 nM. After incubation for 8 h, cells were washed with PBS twice, scraped into 2 mL PBS/EDTA and centrifuged in a 2 mL Eppendorf tube at 500× g. Residual cells in the plate were collected with 2 mL PBS/EDTA, added to the cell pellet and centrifuged again at 500× g. The supernatants were discarded and the cell pellets were stored at −80 °C until further use.
Recombinant tnf α
Manufactured by Merck Group
Sourced in United States
Recombinant TNF-α is a lab reagent produced by Merck Group. It is a cytokine protein that can be used for in vitro research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Growth supplement, by Lonza (1 mentions)
Chondroitinase abc, by Merck Group (1 mentions)
Tnf α, by Merck Group (1 mentions)
Hmvec l, by Lonza (1 mentions)
T75 flask, by Greiner (1 mentions)
Red cell lysis buffer, by Tiangen Biotech (1 mentions)
Lymphoprep, by Takeda Pharmaceuticals (1 mentions)
Ab137321, by Abcam (1 mentions)
Collagenase, by Merck Group (1 mentions)
Ab49900, by Abcam (1 mentions)
Myd88 d80f5, by Cell Signaling Technology (1 mentions)
Sc 7891, by Santa Cruz Biotechnology (1 mentions)
Medium 199 m199, by Lonza (1 mentions)
Roswell park memorial institute medium rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
5 protocols using recombinant tnf α
1
Proinflammatory Cytokine Stimulation of Endothelial Cells
2
Isolation and Priming of Human Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils were isolated as described previously [18 (link)]. In brief, venous human blood for neutrophil isolation was obtained from healthy donors by venipuncture and anticoagulated with ethylenediaminetetraacetic acid (EDTA). Neutrophils were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). Erythrocytes were lysed with ice-cold red cell lysis buffer (Tiangen Biotech, Beijing, China), and then neutrophils were washed in PBS without Ca2+/Mg2+ (PBS−/−; Chemical reagents, Beijing, China) and suspended in PBS−/− to a concentration of 1 × 106 cells/ml and used for further analysis. The trypan blue staining technique was used as an index of the proportion of viable cells in a cell population. Where indicated, cells were primed with 2 ng/ml recombinant TNF-α (Sigma, USA) at 37 °C for 15 min, and untreated cells were incubated with control medium under the same conditions.
3
TNF-α Muscle Damage Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult C57/BL6 mice (8 weeks old; ∼20 g) were injected intraperitoneally with recombinant TNF-α (Sigma; Taufkirchen, Germany; 100 ng/g body weight; n = 7) or with an equal volume of physiological saline solution (n = 6). Twenty-four hours after the injection, mice were sacrificed, and the quadriceps muscle was removed and immediately frozen in liquid nitrogen for subsequent molecular analysis.
4
Evaluating Probiotic Immunomodulation in Caco-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To test the immunomodulatory effects of the tested strains, we designed a preventive model co-culture experiment. First, for the live strains, we centrifuged the bacterial cultures (all strains used in this study, including the pathogens) at 12,000× g for 2 min, washed them twice with PBS buffer, and finally, suspended the pellets in MRS medium for Lactobacillus strains and in meat broth for C. difficile. We used 1 µL of 0.5 OD600 culture for each 300 µL culture medium in each well of the 12-well plate. For the extract fractions, we used 1 µL of 10 μg/µL. We incubated Caco-2 cells with the live Lactobacilli for a total of 18 h. For the pure peptide, we paired this design with another curative model in which incubation with the peptide was allowed after a 2 h challenge with the pathogen, or 1 μL of recombinant TNF-α (20 ng/mL, Sigma-Aldrich, St. Louis, MO, USA) or toxin A (10 ng/mL, Sigma-Aldrich, St. Louis, MO, USA). The experiment was conducted using antibiotic-free medium. The negative control experiments included untreated cells or cells treated with media blanks for the strains, or with DMSO for the fractions, while the positive control comprised cells treated with the inducers only. The choice of the duration of each incubation was based on prior optimization experiments and previous reports on the optimum timing for cytokine release and measurement in Caco-2.
5
Antibody Validation for Inflammatory Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody against ICAM-1 (sc-7891) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against VCAM-1 (E1E8X) and MyD88 (D80F5) were obtained from Cell Signaling, Inc. (Beverly, MA). Antibody against TLR2 (NB100-56728) was obtained from Novus Biologicals, LLC (Centennial, CO). Antibodies against β-actin (ab49900) and GAPDH (ab9385) were obtained from Abcam (Cambridge, MA). Antibody against vimentin (ab137321) was obtained from Abcam (Cambridge, MA). Medium 199 (M199) was obtained from Lonza (Walkersville, MD). Roswell Park Memorial Institute medium (RPMI 1640) was obtained from GIBCO Laboratories (Grand Island, NY). Pam3CSK4 was obtained from InvivoGen (San Diego, CA). TLR2 inhibitor CU CPT 22 (4884) was obtained from Tocris Bioscience (Bristol, UK). Tumor necrosis factor-α (TNF-α) neutralizing antibody (MAB 610) was purchased from R&D System (Minneapolis, MN). Recombinant TNF-α, collagenase and other reagents were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO).
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!