The overall goal of the following experiments is to observe inflammasome activity in in vitro human dendritic cells using simple IL one beta readout assays. First add R 8 48 to the cells to induce intracellular pro IL one beta expression. Then add nige to activate NLRP three inflammasome formation.
That in turn triggers the cleavage of pro IL one beta into mature IL one beta before secretion. Next, collect the samples and measure the intracellular pro IL one beta and the extracellular mature IL one beta results obtained by measuring the secretion of IL one beta from primed and activated cells by immunofluorescence, western blot and eli. A detection demonstrate that priming human DCS results in the intracellular pro IL one beta production in R 8 48 primed cells and secretion of IL one beta from cells both primed and activated with Nigerian juris.
This method can provide insight into the role of the NLRP three inflammasome in human dendritic cell response to synthetic ligands. It can also be applied to other systems designed to test physiological triggers such as bacteria, virus and autoinflammatory disease. Aliquot 200 microliters of freshly isolated monocyte derived dendritic cells in their resting state in a 96 well round bottom plate.
Start with the four standard conditions of unstimulated negative control, priming only, activation only, and priming followed by activation. Then according to experimental design, include diluent controls for R 8 48 and nissin for downstream intracellular cytokine staining assays include duplicates for the isotype controls to prime the inflammasome. ADD R 8 48, add a 10 micromolar final concentration to the appropriate wells.
Place the cells in an incubator at 37 degrees Celsius and 5%CO2 for 18 hours. Then to activate the NLRP three inflammasome, add nige at a 20 micromolar final concentration. Return the cultures to the incubator for an additional six hours to harvest the samples centrifuge the culture plate at 974 times G for three minutes without disturbing the cell pellets.
Transfer each supernatant to a separate round bottom plate in order to measure cytokine secretion by EISA. Now wash the cellular pellets three times with 200 microliters of one XPBS to remove any extracellular IL one beta from the cellular samples for further analysis by a variety of techniques for western blot detection of pro IL one beta lyce the cells directly in 10 microliters of denaturing lysis buffer. After transferring the lysates to 1.5 milliliter einor tubes, heat the samples for 10 minutes at 100 degrees Celsius for fluorescent detection by flow cytometry of IL one beta.
Add 100 microliters of 5%PHS media to the cellular samples and one microliter of fluorescently labeled antibodies to the phenotype markers or isotype control incubate for 10 minutes at room temperature in the dark. After three PBS washes, fix the cells with 100 microliters of 4%PFA for 20 minutes at room temperature in the dark. Next, add 100 microliters of permeable buffer for 30 minutes, followed by 62 nanograms of anti IL one beta FSE antibody or isotype control incubate samples for two hours at 37 degrees Celsius.
Wash the cells three times with 200 microliters of permeable buffer and resuspend in one XPBS. Wrap the samples in foil and store at four degrees Celsius. If detecting IL one beta by microscopy stain cells with dappy, add mountain and place a cover slip gently on top of a glass slide.
Allow the mountain to cure overnight before capturing microscopy images for fluorescent detection by flow cytometry. Acquire data one sample at a time. Set the forward and side scattered gates on the live cells Next gate on the CD 11 C positive CD 14 negative cells.
Finally analyze the MODC population based on pro IL one beta staining in microscopy data acquisition. Set the exposure time with the positive staining R 8 48 treated sample. Then determine the percentage of pro IL one beta expressing MO dcs ease for western blot detection.
Load the total sample volume onto the poly acrylamide gel. Run at 140 volts until the die front runs off the of the gel. Transfer the protein from the poly acrylamide gel onto A-P-V-D-F immo on FL membrane.
Block the membrane with 5%BSA in TBST for one hour. Incubate with the primary antibody while shaking at four degrees Celsius for overnight. After three five minute TBST washes incubate with the secondary antibody at room temperature for one hour after three more TBST washes image the membrane when measuring secreted cytokines by ELI SA equilibrate the samples at room temperature.
Spin down samples to consolidate the supernatant condensation and follow the manufacturer's instructions for measurement of IL one beta intracellular cytokine staining for pro IL one beta allows for microscopy and facts readouts from CD 11 C positive CD 14 negative monocyte derived dendritic cells. Both techniques can be quantified relative to a non-prime or resting cell control as well as an isotype control. The percent of pro IL one beta stain cells is multiplied by the geometric median of this population to provide the median fluorescent intensity.
The MFI is comparable to the amount of pro IL one beta present in the positive stain cells.Here. Immuno blotting techniques are used to measure pro IL one beta from the cell lysates. Quantitative data is expressed relative to an internal cellular control such as beta tubulin as expected.
Immuno blotting for pro IL one beta in NI Nigerian treated cells reveals a decrease in pro IL one beta. This is complimented by a concurrent increase in IL one beta in supernatants, measured by E ELI a only an R 8 48, followed by NI Nigerian conditions. Simultaneous measure of other inflammatory cytokines such as T NF alpha IL 10, and IL six ensure that Niger is specific in causing the secretion of IL one beta.
The level of priming is time and dose dependent. Further experimentation such as measuring ex extracellular protein concentration, chemiluminescence detection from mature I one beta generation or blocking the inflammasome would help determine whether ex extracellular IO one beta is the mature cleaved form and whether IO one beta secretion is nlrp. Three inflammasome activity dependent.
