Starting in mid July , 2020 we are going to provide providing the cytokine multiplex assay as a new service. This assay is validated for serum , plasma and culture supernatant.

With as little as 12.5 µl serum/plasma, or 25 µl for tissue culture samples per well, this panel allows simultaneous quantification of 13 12 human cytokines:

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Details of the kit can be found here:

|https://www.biolegend.com/en-us/products/legendplex-humanhu-th-cytokine-panel-1312-plex-filterwfp-platev02-1579119471

Brief protocol:

1. Thaw samples on ice. Centrifuge @ 10000 g for 10 min at 4^oC12000g for 5min.
2. Dilute 50 ul of serum samples 1:1 with assay buffer. Cell culture supernatant samples can be tested without dilutions but may need a preliminary experiment to determine the appropriate dilution factor.
3. Prepare 1:4 serial dilution of standards.
4. Pre-wet filter plate by adding dispensing 100 ul of wash buffer . Vaccumusing the plate washer. Vacuum.
5. Add 25 ul of assay buffer to each well. When pipetting aim for the side of the wells and avoid touching the membrane. This can help preventing leakages.
6. Add 50 ul of diluted sample/standards & 25 ul of mixed beads to each well.
7. Seal plate and shake at 500 rpm for 2 hours RT.
8. Wash with dispensing 200 ul of wash buffer using the plate washer and vaccumvacuum.
9. Add 25 ul of detection antibodies to each well. Seal plate and shake at 500 rpm for 1 hour RT.
10. Add 25 ul of SA-PE to each well. Seal plate and shake at 500 rpm for 30 min RT.
11. Wash with dispensing 200 ul of wash buffer and vaccumusing the plate washer and vacuum.
12. Add 150 ul of wash buffer to each well. Resuspend beads on a plate shaker for 1 min.
13. Samples are now ready to read on a cytometer.

Cost:

Each sample will cost $40~$50-58 (subject to kit/sample prep). We will be running 39 samples 40 samples (including 1 assay control) plus 8 standard curve titrations in duplicate on a 96 well plate.

We'll also include a frozen aliquot of sample standard to monitor variation between plates.

To order this service, please complete an order for Service_Cytokine Assay under the Flow Cytometry tab on PPMS:

https://au.ppms.info/wrh/order/?pf=2

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