PBMCs of all participants were thawed, washed and put to rest for 1 h in RPMI+glutamax with 10% FBS in a 96-wells round-bottom plate (Greiner), at a concentration of 200.000 PBMCs/well. After resting, the PBMCs were stimulated with one stimulus per well for 24 h at 37 °C, 5% CO2. Stimuli used were LPS EK (E.coli K12 ultrapure) 10 ng mL− 1 (TLR4), CpG ODNM362 10 μg mL− 1 (TLR9), and R848 10 μg mL− 1 (all Invivogen) (TLR7&8). After stimulation, the supernatants were collected and stored at − 80 °C for later use. The supernatants were thawed and analyzed in two batches on a FACSCanto™ flow cytometer (BD Biosciences), using a custom-made bead-based immunoassay (LEGENDplex™, BioLegend) according to the manufacturer’s instructions, for the following cytokines and chemokines: sGP130, IL-6, IFNγ, IL-10, IL-1β, TNFα, MCP-1, RANTES, CXCL8/IL-8, CXCL10/IP10, and IFNα. In addition, concentrations of sIL6R were quantified using a commercially available ELISA kit (R&D Systems).
Cpg odn m362
Manufactured by InvivoGen
Sourced in United States
CpG ODN M362 is a synthetic oligodeoxynucleotide (ODN) that contains CpG motifs. CpG ODNs are known to activate the immune system by binding to Toll-like receptor 9 (TLR9).
Automatically generated - may contain errors
Lab products found in correlation
Legendplex, by BioLegend (1 mentions)
Lps ek, by InvivoGen (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps from e coli k12, by InvivoGen (1 mentions)
Lipoteichoic acid lta from s aureus, by InvivoGen (1 mentions)
Malp2, by Alexis Biochemicals (1 mentions)
Mouse cd40 ligand, by R&D Systems (1 mentions)
24 well culture dish, by Sarstedt (1 mentions)
4 protocols using cpg odn m362
1
Stimulation-Induced Cytokine Profiling
2
PBMC Stimulation and mRNA Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were thawed and stimulated with 1 ng/ml lipoteichoic acid (LTA) from S. aureus (InvivoGen, San Diego, USA), 1 ng/mL lipopolysaccharide (LPS) from E. coli K12 (Invivogen) or 1 μg/ml CpG ODN M362 (InvivoGen) in AIM V (Life Technologies AB, Täby, Sweden) with 20 μM mercaptoethanol (Sigma-Aldrich) at a concentration of 1 × 106/ml and incubated for 24 hours at 37°C. Unfortunately, enough cells were not always available for all stimuli. Cell cultures were then centrifuged, supernatants were collected and cell pellets were lysed in RLT-buffer (Qiagen GmbH, Hilden, Germany) and stored at −70°C for later mRNA analysis. The mean viability of the freeze/thawed PBMCs was 85.2%.
3
Immune Modulator Reagent Acquisition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CpG (ODN M362) was purchased from Invivogen (United States), MALP-2 from Alexis Biochemicals (United States), and LPS from Salmonella minnesota from Enzo Life Sciences GmbH (Germany). All chemicals used were of analytical grade and received from commercial sources.
4
Trophoblast-Induced IL-10-Producing B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Swiss Webster mouse trophoblastic cell line SM9-2, kindly provided by Joan Hunt and David Wheaton (University of Kansas Medical Center, Kansas City, MO, USA), was cultured in RPMI 1640 medium containing 20% charcoaled fetal bovine serum (FBS), 2 mM l -glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 1% penicillin–streptomycin (P/S). Cells were seeded at a concentration of 5 × 104 cells/well on a 24-well culture dish for 24 h (Sarstedt, Newton, NC, USA). Trophoblast attachment to the culture dish was assessed under the microscope, and old medium was carefully removed. To study the effect of trophoblast-derived factor(s) on the generation of IL-10-producing B cells, 500 μl of fresh medium containing 1 × 105 CD19+ eGFP− B cells were added to SM9-2 cells at ratio of 2:1 for 24 and 48 h in the presence or absence of mouse CD40 Ligand (R&D Systems, Minneapolis, MN, USA) and CpG ODN M362 (InvivoGen, San Diego, CA, USA). Cell expression of GFP was analyzed by flow cytometry and corresponded to IL-10 production. The 0.5–1 × 105 events were measured in each case.
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!