The overall goal of the following experiment is to predict the prognosis of idiopathic scoliosis according to the functional grouping of patients by screening their PBM C'S response to GPCR R activation using cellular dielectric spectroscopy. This is achieved by culturing pbmc in the presence of phyto hemagglutinin or PHA to expand cell number. Cell expansion is completed in a tube, maintained in an inclined position.
As a second step, the cells are treated with OSTEOPONTIN or OPN, which increases or reduces GI protein activity. Depending on the patients after treatment, the cell suspension is transferred into the microplate. Next, the cells are stimulated with GI or GS GPCR agonists to determine the resulting impedance changes.
The results provide kinetic responses for all wells and integrated responses are then quantified based on impedance changes. The implication of this technique is well beyond simply diagnosis. So for the first time, we can detect at a early stage as symptomatic patients at risk of developing scoliosis, and now knowing who will develop scoliosis or at what stage they are, we can provide new therapies to treat them.
The main advantage of this technique over existing methods like x-ray exposure, is that this technique allows predicting progno osteopath scoliosis with no need to measure covid and no health risk. The most recent thing, the technique now will be our research assistant Nita Franco, Thaw of frozen aliquot of peripheral blood mononuclear cells in a 37 degree Celsius water bath. Using a sterile pipette transfer the cell suspension to a 50 milliliter tube containing 15 milliliters of complete media and centrifuge.
The cells aspirate the supernatant. Then gently suspend the cell pellet in one milliliter of PHA media, complete the volume to 20 milliliters with the same media cap the tube loosely to allow air to enter. Incubate the tube overnight at 37 degrees Celsius in a carbon dioxide incubator to allow quiescent lymphocytes to transform into rapidly proliferating lymphoblasts.
Tighten the caps completely and pellet the cells by centrifugation. Remove the supernatant by aspiration. Gently suspend the cell pellet in one milliliter of complete media.
Complete the volume to 20 milliliters with the same media and cap the tube loosely culture overnight to expand the cell numbers. After two washes with 10 milliliters of RPMI 1640, gently resuspend the cell pellet in 600 microliters of RPMI 1640. Measure the cell concentration and viability using an automated cell counter and viability analyzer.
Then add an appropriate volume of RPMI 1640 to adjust to a cell concentration of 1.5 times 10 to the fifth cells per 20 microliters. Transfer 100 microliters of the suspension to two sterile 1.5 milliliter eend orph tubes. Add recombinant osteopontin in one tube to a final concentration of 0.5 micrograms per milliliter.
Add an equal volume of PBS to the second tube. Gently mix each condition by pipetting twice using a sterile pipette set at 100 microliters. Next, add five microliters of RPMI 1640 to each well of a 96 Well plate centrifuge the plate at 200 GS for three minutes to remove any air bubbles to transfer the cells from tube to microplate.
Gently pipette up and down once to ensure a uniform suspension of cells. Seed 40 microliters of cell suspension per well in quad duplicate for untreated cells and in duplicate for ROPN or PBS treated cells. Leave the cell plate under the sterile hood for five minutes to allow cells to rest and settle evenly to the bottom of the well.
To optimize the effect of OPN culture, the cells for 18 hours at 37 degrees Celsius in a carbon dioxide incubator equilibrate the cultures at room temperature for around 30 minutes. Meanwhile, prepare one milliliter of 100 micromolar somatostatin and isoproterenol in RPMI 1640. Fill the compound plate by dispensing 20 microliters in appropriate wells.
Cover the compound plate with a curable seal. Load the plate pipette tips and compound plate into the CDS based system. Name the plate in the CDS based instrument software.
Go to the protocol list and select the appropriate protocol to initiate the protocol. Click start. The integrated fluidic system simultaneously adds the compounds to all wells.
The CDS based system automatically collects the data for 15 minutes after compound edition cell viability was comparable between all samples with values consistent in the range of 86 and 96%In contrast, high variations were noted in cell numbers. Among samples of the 32 samples, used two had an insufficient number of cells and have not been classified. This example shows the functional classification according to the degree of imbalance between GI and GI signaling.
The evaluation of the OPN effect in response to GI stimulation had revealed that OPN increased the response in patients 353 and 371. In contrast, the response was reduced by more than 50%in patients 345 and 382 and by less than 50%in patient 370 following ROPN treatment. So according to our classification criteria, we were able to categorize patients 353 and 371 in FG one patients 300 from 45 and 382 in FG two and patient 370 in FG three.
In parallel, all patients were screened for their response to GI protein stimulation and compared to control subjects. As expected, all patients were less responsive than control subjects. Patients classified in the same functional group exhibited similar levels of the maximum response.
The classification of a large cohort of SC patients regularly followed in our special clinic at San Justine Hospital has revealed that the three functional groups were similarly distributed. Among moderate cases, the FG two was predominant among severe cases. After waiting this video, you should have a good understanding of how to classify patient in two, three functional groups using cellular dielectric spectroscopy.
