Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using Ficoll-Pague Plus (GE Healthcare). PBMCs were cultured in RPMI 1640 supplemented with 5% (v/v) fetal calf serum (Gibco), 2 mmol/L l -glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at a density of 1 to 1.5×106 cells per well in 48-well plates. Cells were stimulated with 1 µg/mL phytohemagglutinin M (Calbiochem) in the presence or absence of different HDL preparations and incubated for 20 hours at 37°C in a CO2 incubator. To prevent potential effects of endotoxin contamination, cell culture medium was supplemented with 1 μg/mL polymixin B (Sigma-Aldrich); HDL and phytohemagglutinin M were preincubated with polymyxin B for 15 minutes before being added to PBMC. Tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6, and macrophage inflammatory protein-1β were measured in cell-free supernatants using Human Cytokine Magnetic 4-Plex Panel (R&D Systems).
Phytohemagglutinin m
Manufactured by Merck Group
Phytohemagglutinin M is a laboratory reagent derived from the red kidney bean (Phaseolus vulgaris). Its core function is to act as a mitogenic agent, stimulating the division and proliferation of lymphocytes, particularly T cells, in cell culture and research applications.
Automatically generated - may contain errors
2 protocols using phytohemagglutinin m
1
Cytokine Profiling of PBMC Responses
2
Evaluating LAG-3 Targeting Probes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immune cells from C57BL/6 mice and human peripheral blood mononuclear cells (hPBMCs; catalog no. HPA01091; Hope Biotechnology) were used. Immune stimulation with an activation cocktail (catalog no. 423301; BioLegend) on C57BL/6 mouse-derived immune cells and phytohemagglutinin-M (catalog no. 11082132001; Sigma) on hPBMCs was performed for flow cytometry analysis of LAG-3 and CD8. Cell uptake of [ 68 Ga]Ga-NOTA-C25 or [ 68 Ga]Ga-NOTA-scrambled C25 (0.037 MBq/mL; 6.788 3 10 29 mM) was performed by blocking assays with nonradiolabeled C25 (9.613 3 10 26 mM) and anti-LAG-3 antibody (aLAG-3; 1.333 3 10 27 mM) to test probe specificity. Detailed protocols are provided in the supplemental materials.
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!