The overall goal of this protocol is to study the cytokine profiles in human tear samples collected by Schirmer Strips using a bead-based multiplex assay. This method can help answer key questions in the field of ophthalmology such as the role of cytokines in various ocular diseases. The main advantage of this technique is that it can be used to study multiple analytes in smaller sample volumes.
Demonstrating the procedure will be Praveen Balne, a post-doc from my lab. After collecting tears from a subject according to the text protocol, cut the tear flow strip to a length of 0.5 centimeters and place it in a sterile 1.5 milliliter microcentrifuge tube. Add 30 microliters of assay buffer and incubate the tube at room temperature for five minutes.
Then centrifuge the tube at 14, 000 times g for one minute. Transfer the supernatant into another 1.5 milliliter microcentrifuge tube and discard the strip. Then place the tube containing the sample on ice and use the eluted tear sample immediately for cytokine profiling.
To carry out the bead-based multiplex assay, bring all reagents to room temperature and vortex them for five to 10 seconds. If eluted tear samples were stored at minus 80 degrees Celsius prior to the assay, thaw the frozen tear extracts on ice and centrifuge the samples at 1, 000 times g for five minutes. Prepare an assay worksheet in a vertical configuration for working human cytokine standards QC-1, QC-2, and samples.
Add 200 microliters of 1X wash buffer to each well of the plate and seal it with a plate sealer. Then incubate the plate on a plate shaker at room temperature for 10 minutes. Next, decant the 1X wash buffer by inverting the plate and tap onto absorbent towel several times to remove any residual amount of wash buffer in the wells.
Then add 25 microliters of each working human cytokine standard QC-1, QC-2, the blank with just assay buffer, and samples into the appropriate wells. Add 25 microliters of assay buffer into each well. Then to the wells, add 25 microliters of 1X antibody bead cocktail solution.
As the antibody bead solution is light sensitive, seal the plate and cover it with aluminum foil and plate cover to protect from light during the assay. Incubate the plate at four degrees Celsius on a shaker overnight. The following day, place the plate on the plate rack of an automatic magnetic plate washer and let it sit for one minute to settle the magnetic beads at the bottom of the well.
Aspirate the well contents and add 200 microliters of wash buffer per well using automatic magnetic plate washer. Let the plate sit for one minute and then aspirate the well contents as before and repeat the wash one more time. Add 25 microliters of detection antibody solution into each well.
Seal the plate. Cover it with aluminum foil and plate cover and incubate it at room temperature on a shaker for 60 minutes. Then add 25 microliters of Streptavidin Phycoerythrin solution into each well and after sealing and covering the plate, incubate it at room temperature on a shaker for 30 minutes.
Following the incubation, place the plate on a magnetic plate washer and let it sit for one minute. Then aspirate the well contents and add 200 microliters of wash buffer per well. Let it sit for one minute then aspirate the well contents before repeating the wash once again.
Add 150 microliters of sheath fluid to each well. Then place the plate on a shaker for five minutes at room temperature to resuspend the antibody beads before proceeding to read the plate and analyze the cytokine concentrations. To read the assay plate according to the text protocol, open the batches page and click create a new batch tab from an existing protocol.
In the batch name box, type the batch name and type a description about the batch in the enter optional description box. Click on the previously generated protocol then click next. The next tab that opens is the standards and controls tab.
View the details of the assay standards and select next. Under the plate layout tab, assign well commands for the batch and click save to save batch information to the pending batch list. Load the plate into the bead-based multiplex assay plate reader and run the batch from the pending batch list.
Launch the RBX conversion software and under select exponent files, select the csv file generated from the bead-based multiplex assay software. Then click on the generate button. A rbx file will be saved in the selected output folder.
Now, launch the analysis software and open the rbx file. Then using the 4PL-5PL curve fit, optimize the standard curves. To optimize the standard curves, select the following in the standard curve tab, regression type, logistics 5PL, and access transformation log X, linear Y.Then tick the following boxes, show concentration range lines, show unknown samples, show control samples, apply across all analytes, and show report after optimization.
Click the optimize button for auto optimization. Under the enter sample info tab, label the sample identities. Finally, obtain the observed concentrations in the report table tab and click on export report table to generate the data in a spreadsheet file for further analysis.
Tear samples were collected from eight eyes of four healthy men ages 36 to 52. Of the 41 cytokines analyzed using the bead-based multiplex assay, 31 were detected in all tear samples. For example, IL-17A, MIP-1b, and TNFa were detected in seven eyes of four subjects and IL-4 was detected in six eyes of four subjects.
In addition, interferon gamma was detected in six eyes of three subjects. Interleukin-1a was detected in five eyes of three subjects. Eotaxin and Interleukin-9 were detected in three eyes of two subjects.
Interleukin-3 was detected in one eye and MIP-1a was detected in none of the samples. The mean plus or minus standard deviation concentrations of the 41 cytokines detected are illustrated in this scatter plot. Interleukin-1ra was highly expressed in all the samples.
Cytokines IP-10, GRO, MCP-1, PDGF-AA, Fractalkine, Interleukin-a, EGF, PDGF-BB, VEGF, and G-CSF were also highly expressed at nanogram per milliliter levels and the remaining cytokines were expressed at picogram per milliliter levels. As seen here, the lowest measured concentration of tear cytokine was TNF-a with a mean plus or minus standard deviation of 27.25 plus or minus 19.97 picograms per milliliter. In 31 cytokines which were detected in all the samples, none showed a statistically significant difference in concentration between the right and left eye by T test.
Once mastered, this technique can be done in 1-1/2 days if it is performed properly. After watching this video, you should have a good understanding of how to perform a bead-based multiplex assay for tear cytokine profiling.
