The overall goal of this procedure is to measure pro-inflammatory cytokines produced in a whole blood stimulation assay. First, the cellular components of whole blood are isolated from the plasma by centrifugation, and the white blood cells are enumerated using aine orange staining. Next, the cells are plated and stimulated with LPS followed by a TP to elicit cytokine production by cells bearing microbial pattern recognition receptors cultured supernatants are then collected at specific time points following stimulation initiation.
Finally, cytokine concentrations in the supernatants are measured in a 96 well plate multiplex assay. Using this simple method, variations in the concentrations of pro-inflammatory cytokines produced by stimulated cells from different donors can be rapidly assessed. This method is especially useful to identify I one mediated inflammatory disease in patient presenting with pathologically antiinflammatory manifestation, particularly under circumstances where the volume of blood attainable is limited.
The main advantage of this technique over existing methods when measuring cytokine concentrations is that the luminex based technology allows the simultaneous measurements of several cytokines while only using a small sample volume. Obtain samples containing 10 milliliters or more of venous blood from affected individuals, as well as from healthy age and gender matched controls ensure that the samples are collected in sodium heparin, vacutainer blood collection tubes, and the EDTA or other anticoagulants are not added as these will interfere with the assay and process the samples as soon as possible after collection. If necessary, the samples can be stored for up to 24 hours at room temperature under dim lighting conditions.
Centrifuge the blood samples at 3000 RPM for 10 minutes at room temperature with the break off. Once samples have been centrifuged, remove the plasma from the top of the tubes being careful not to disturb the buffy coat layer containing mononuclear cells. Resus, suspend the buffy coat and the underlying red blood cell pellet in room temperature serum free RPMI medium to wash the cells gently pipetting the mixture.
Then transfer the cells from the collection tube into a 50 milliliter conical. Top up the volume to 50 milliliters with RPMI and gently invert each tube to mix. Centrifuge the cells once more at 3000 RPM for 10 minutes.
Then aspirate off the supernatant, being careful not to disturb the cell pellet. Repeat this wash step two to four times in order to remove contaminants and eliminate cell bound cytokines once the cells have been washed, resus suspend with serum free RPMI to a total volume of 20 milliliters. To count the cells load 20 microliters of a one-to-one mixture of sample and aine orange In a disposable soter vision chamber, Aine orange used at a final concentration of one microgram per milliliter will stain only nucleated white blood cells, allowing them to be differentiated from red blood cells.
Count only the D positive cells once a cell count has been obtained. Re suspend the samples in serum free RPMI so as to attain a final concentration of 2 million cells per milliliter. For each sample labeled duplicate wells of a 24 well plate with the experimental conditions to be tested.
In this case, the conditions are unstimulated, LPS alone, a TP alone, and LPS plus A TP.Add one milliliter of diluted cells to each well and add LPS to the appropriate wells at a final concentration of one microgram per milliliter. Incubate the samples at 37 degrees Celsius in a 5%carbon dioxide incubator for two hours and 40 minutes. After this initial incubation period, add the a TP at a final concentration of two millimolar in the A TP only and LPS plus a TP.Wells then incubate the plates for a further 20 minutes at 37 degrees Celsius.
Once the second incubation is complete, transfer the cells from each well into a separate pre-labeled 1.5 milliliter micro centrifuge tube, centrifuge the samples at 10, 000 RRP M for two minutes at room temperature, then transfer the supernatant from each tube into a new pre-labeled EOR tube. Supernatant can be assayed for cytokines immediately or stored frozen to assess concentrations of IL one beta or other cytokines. In the samples, we use the bio plex pro, human cytokine X plex with the Bio Plex 200 system.
According to the manufacturer's instructions. In this figure analysis of IL one beta production upon stimulation of normal whole blood cells with L-P-S-A-T-P or both stimuli together shows a response LPS plus A TP, the magnitude of which is dependent on the number of cells being stimulated. In contrast to healthy controls whole blood assays from patients displaying autoinflammatory pathologies reveal enhanced IL one beta production following LPS stimulation in the absence of exogenously added a TP.Don't forget that working with human body fluids such as blood can be extremely hazardous and universal precautions should always be taken when performing this procedure.
