Need to detect cytokines from PBMCs dosed with peptides. This is to determine if the peptides elicit PBMCs to release markers for immune response. I am unsure how long to incubate the PBMCs with the peptides? And.. Do you need to dose the cells multiple times?
"Need to detect cytokines from PBMCs dosed with peptides. This is to determine if the peptides elicit PBMCs to release markers for immune response."
- What kind of peptides, which cytokines? Human or which species?
"I am unsure how long to incubate the PBMCs with the peptides? And.. Do you need to dose the cells multiple times?"
- Depending on the treatment, what do you want to see? What is your hypothesis? Your question is quite unspecific.
1. Question/hypotheis
2. species
3. cytokines
4. stimulating peptides
- Bacterial peptides (15-mers and 9-mers)
1. Question: Will peptides of our bacterial transgene elicit an immune response if expressed in engineered human cells? As a preliminary experiment peptides of the bacterial gene will be exposed to human PBMCs to determine if the bacterial protein is immunogenic.
2. E. coli protein
3. INFg and TNFa
4. 9-mers and 15-mers of the E.coli protein
Hello!
You can look at this protocol:
4.4. Ex-Vivo Induction of Cytokines from rhIL-1β and LPS-Stimulated PBM
The PBMC were placed in 24-well plates after the density adjustment at 1 × 106 cells/mL (final volume of 1000 µL). Cells were cultured in RPMI-1640 medium without serum and supplemented with antibiotic and antifungal. Untreated cells were taken as the control group for each experiment. The effect of LPS (30 ng/mL) in the secretion of TNF-α was studied in samples from four HBD to demonstrate the positive stimulation of the cells
LPS response in PBMC was determined at 0, 10, 30 and 50 µM. Subsequently, PBMC culture was performed with LPS (30 ng/mL). Each experiment was done with two biological replicates and was incubated for 24 h at 37 ◦C in a humidified 5% CO2 atmosphere.
4.5. Cytokine Analysis by Multiple Analyte Profiling (xMAP) Technology The concentration of IL-1β, TNF-α, IFN-γ, IL-6, IL-12, IL-17A, M-CSF, GM-CSF, IL-2, VEGF, IL-15, IL-7, IL-1Ra, IL-4, IL-5, IL-13, IL-10 and IL-9 and were quantified from culture supernatants by multiplex immunoassay method using the Bio-Plex ProTM Human Cytokine 27-Plex Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. Luminex MAGPIX® (Luminex Corporation, Austin, TX, USA) instrument was used for xMAP assays
Or
2.3. Whole Blood Stimulation Heparinized whole blood was taken from healthy rats and added in 96-well plates (200 μL/well), supplemented with different concentrations of each stimulating reagent (PMA: 1, 5, and 25 ng/mL; Ionomycin: 1 μg/mL; PHA, LPS, Con A, PWM: 1, 5, and 10 μg/mL), and incubated for 0, 2, 4, 6, 8 or 10 h at 37 °C with 5% CO2.
Alexandr Chernov thank you for your response! For the protocol that you suggested, you measured cytokines at relatively short time points. Is PBM cell proliferation necessary to show a measurable and/or biologically significant amount of cytokine release? From the time points that your protocol uses, it would indicate no.
Hi,
There are several methods for identifying antigen (peptides here)-specific T-cells e.g. proliferation (measured after several days of cell culture stimulation), intracellular cytokines staining, etc. But these methods are classically used when memory antigen-specific cells are present. If I understood well, in your case using neo-antigens in-vitro, only naive cells would be potentially present. This means that you should focus on methods designed to identify naive cells potentially responding to these neo-antigens, which will demand several days of culture in the presence of cell-growth cytokines (such as IL-2). By using tetramers it is possible to identify ex-vivo naive antigen-specific T-cells but limited to HLA-peptide specific combinations. Maybe it would be interesting to immunize mice with your candidate peptides and look for peptide-specific T-cells eventually elicited? I am not familiar with B-cells but I suppose the problem would be similar.
Few effective answers here but hope it helps to target the best approach...
Marcelo De Carvalho Bittencourt Thank you for your thoughtful reply. This is helpful information. I think we are most interested in a relatively high throughput assay so that we can get some quick information about what peptides of our E.coli protein of interest could be immunogenic to immune cells. I think the mouse experiment you suggested is of interest to us but at a later stage. Thanks again.
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