Bio-Rad Bio-Plex Pro™ Human Th17 Cytokine Assays User Manual

Page 20

Table 8. Detailed wash and incubation instructions.

Add Coupled Beads, Samples, Standards, Blank,

and Controls

1. Cover unused wells of the assay plate with sealing tape.

2. Prewet the filter plate. Skip this step if using a flat-bottom plate.

a) Vacuum manifold: Prewet the wells with 100 µl assay buffer and

remove the liquid by vacuum filtration. Dry the bottom of the filter
plate thoroughly by blotting on a clean paper towel.

WASH

After each assay step, select the appropriate Bio-Plex Pro

™

wash station program, or perform the

appropriate manual wash step as summarized below.

Using the Bio-Plex Pro Series Wash Stations

For detailed instructions, please refer to the quick guide (bulletin 5826) or user manual (item

#10013125). Both documents can be found at www.bio-rad.com/bio-plex.
Using the Bio-Plex

Handheld Magnetic Washer

For detailed instructions, please refer to the user guide (item #10023087), which can be

found at www.bio-rad.com/bio-plex.
Using a Vacuum Manifold

Place the filter assay plate on a calibrated vacuum apparatus and remove the buffer by

vacuum filtration. Add 100 μl wash buffer to each well and remove the liquid as before.

Ensure that all wells are exposed to the vacuum. As a general guideline, 100 μl should take

approximately 3–4 sec to completely clear the well. Repeat as specified. Thoroughly blot

the bottom of the filter plate with a clean paper towel between each vacuum step to prevent

cross contamination and plate leakage. Place the assay plate on the plastic plate holder.

COVER AND INCUBATE
Gently cover the plate with a new sheet of sealing tape. To prevent cross-well contamination

or leaking from the bottom of filter plates, do not press down on the tape. Place the plate on a

microplate shaker and cover with aluminum foil to prevent photobleaching. Vigorous shaking

is important. Shake at room temperature at 850 ±50 rpm for the specified incubation time.

Slowly ramp up to the maximum speed to avoid splashing of samples.

Bio-Plex Pro or

Bio-Plex Pro II

Handheld Magnet or

Pro II Wash Station

Wash Station

Vacuum Manifold

Assay Step

Magnetic Program

Vacuum Program

Manual Wash Steps

Add beads to plate

MAG x2

VAC x2

2 x 100 μl

Sample incubation
Detection Ab incubation

MAG x3

VAC x3

3 x 100 μl

SA-PE incubation