Bioplex Cytokine
assay:
Purpose:
- to quantitate multiple cytokines in various samples including serum, plasma, and tissue culture supernatants.
-
it incorporates novel
technology using color-coded beads, permits the simultaneous detection of
multiple cytokines in a single well of a 96-well microplate.
It consists of 3 core technologies:
I. Family of fluorescently dye microspheres (beads) to which biomolecules are bound
II. Flow cytometer with two lasers and associated optics to measure biochemical reactions that occur on the surface of beads
III. High-speed digital signal processor that efficiently manages the fluorescent output
Principle:
It is designed in a capture sandwich immunoassay format. Antibody specifically directed against the cytokine of interest is covalently coupled to color-coded 5.5µm polystyrene beads. The antibody coupled beads are allowed to react with a sample containing an unknown amount of cytokine, or with a standard solution containing a known amount of cytokine.
A biotinylated detection antibody specific for a different epitope on the cytokine is added to the beads. The result is the formation of a sandwich of antibodies around cytokine. The reaction mixture is detected by the addition of Strepavidin-phycoerithrin (Strepavidin-PE) which binds to the biotinylated detection antibodies.
The constituents of each well are drawn up into the flow-based Bioplex suspension array system, which identifies and quantitates each specific reaction based on the bead color and fluorescence. The magnitude of the reaction is measured using fluorescently labeled reporter molecules associated with each target protein. Unknown cytokine concentrations are automatically calculated by Bioplex Manager Software using a standard curve derived from a recombinant cytokine standard.
Procedure:
I use this technique to detect cytokine levels (e.g. IL-6, IL-2β,
TNF-α, IFN-γ, CCL2 (MCP-1), KC, MIP-1α, and Rantes in cornea and
TG (trigeminal ganglion) of WT and CXCL10 knock out (or TLR9 knock out) uninfected
vs infected mice with HSV-1 at day 3, 5, and 7 post-infection.
I harvest the cornea and TG in 250µl and 500µl RPMI-1640 with protease
inhibitor cocktail respectively. Homogenize the sample and centrifuge it to get
at least 220 µl. After getting sample, proceed for following procedure;
1. Prepare cytokine standard (Multiplex cytokine standard) by reconstituting 1 tube of the lyophilized cytokine standard with 500 µl of appropriate matrix (e.g. RPMI medium).
Gently mix by flicking the bottom of the tube 3-5 times and incubate on ice for 30 min. (50,000 pg/ml per cytokine).
2. Prepare cytokine standard curve in following range 0.2- 3,200 pg/ml by serial dilution in RPMI.
3. Calibrate the vaccum filter using 96 well plate and fix the Hg level in between 1 and 2.
4. Make Working bead solution by mixing 5,760 µl of Bio-plex assay buffer and 240 µl of 25X stock beads in a tube covered with aluminum foil and place in dark.
5. Perform Multiplex Assay procedure:
I. Place 150 µl of bioplex assay buffer to pre-wet desired number of wells and remove the buffer by vaccum filtration. Dry the bottom of the filter plate thoroughly with a clean paper towel.
II. Vortex the multiplex bead working solution for 15-20 sec at medium speed and pipet 50 µl into each well. Vaccum filter.
III. Dispense 100 µl of bioplex wash buffer to each well. Remove by vaccum filtration.
IV. Pipet 50 µl standard or sample per well. Change the tip after every transfer. Cover the filter plate with sealing tape.
V. Place the filter plate onto a microplate shaker. Speed the shaker slowly to 1,000 rpm and maintain for 30 sec and reduce to 3,00 rpm for 30 min.
6. Using Detection antibody (50X):
I. Pipet detection antibody diluent A (2940 µl) and place in a tube covered with foil.
II. Add 60 µl of 50X stock detection antibody. Place in dark.
III. Add 25 µl of detection antibody to each well of filter plate (after washing 3X with wash buffer)
IV. Place filter plate onto a shaker after sealing with tape and follow the same rpm (1000 rpm for 30 sec. and 3,00 rpm for 30 min.)
7. Using Strepavidin-PE preparation:
I. Pipet 5940 µl of Bioplex assay buffer in a tube covered with foil.
II. Pipet 60 µl of 100X strepavidin-PE into the tube. Place in dark.
III. Add 50 µl of strepavidin-PE preparation to the filter plate after washing 3X with bioplex wash buffer. Cover the plate with tape.
IV. Place in shaker and follow the same rpm.
V. Incubate for 10 min at room temperature.
VI.
Remove the buffer by vaccum filtration.
VII. Resuspend the beads in each well with 125 µl of bioplex assay buffer. Cover with tape. Place on shaker and shake at 1000 rpm for 30 sec. immediately before reading the plate on bioplex system.
How to use Bioplex
system (The machine):
A. Turning on machine
1. Turn on machine (2 switches at back on bioplex system; 1 switch on Bioplex HTF
system).
2. Turn on Bioplex manager on computer.
3. Add dH2O into one compartment of Bio-rad plate and 70% propanol into another
compartment.
4. Click Eject/Retract and put Bio-rad plate and press Eject/Retract again.
5. After about 5 min, press warm up and press Eject/Retract again and let for warm up.
B. Calibration:
1. If running 0.2-3,200 pg/ml cytokine standard curve 9High RPI target value), need to
calibrate using High RPI (high PMT) calibration for Cal2.
2. Press calibrate, enter name, press Cal1 and Cal2.
3. Match the number given in vial Cal1 and cal2.
4. Press Eject/retract
5. Add 5 drops of cal1 and 5 drops of cal2 to respective place in Bio-rad plate after
vortexing for 30 sec.
6. Press Eject/retract, press ok.
C. Protocol for analysis:
New protocol
Describe protocol
Select analytes
Format plate
Enter standard information
Enter control information
Enter sample information
Run protocol
After running protocol, bioplex manager will give standard curve and value of each unknown cytokine in samples. Analyze the data.