Bio-Rad Bio-Plex Pro RBM Canine Kidney Toxicity Assays User Manual

Bio-Plex Pro Assay Quick Guide

Volume of Volume of SA-PE Dilution SA-PE, µl 1x Assay Buffer, µl Total Volume, µl

1:10 225 2,025 2,250

10. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min a t R T.

11. Wash the plate three times with 100 µl 1x assay buffer.

12. After the ﬁnal wash, resuspend the beads in 100 µl assay buffer. Cover plate as in Step 4 and shake the plate at 850 ± 50 rpm for 30 sec.

Bio-Plex Pro™ RBM Kidney Toxicity Assays

Quick Guide

For Use With Instruction Manual #

Bio-Plex Pro™ RBM Human, Rat, a nd Canine Kidney Toxicit y Assays 100 28258

13. Remove the plate seal and read plate at low PMT (Bio-Plex® 200), standard

PMT (Bio-Plex 3D), or default settings (Bio-Plex® MAGPIX™).

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This guide can be used to prepare and run a full 1 x 96-well assay plate. For more information on a given step, refer to the corresponding section of the complete instruction manual. New users can download the manual, which includes detailed instructions and a list of kit components, at www.bio-rad.com/bio-plex.

IMPORTANT! Pay close attention to vortexing, shaking, and incubation instructions.

Deviation from the protocol may result in low assay signal and assay variability.

A. Reagent Preparation

1. Reconstitute the following lyophilized reagents in dH20 before use

according to the table below.

Reagent Volume, µl Reagent Volume, ml

Standards mix 150 Blocking buffer 1.5

Control 1 100 Standard diluent 1.0

Control 2 100 Detection antibodies 4.8

a. Allow vial to sit at room temperature for a minimum of 5 min, not to

exceed 30 min.

b. Mix by vortexing at a medium setting.

2. Bring the 10x assay buffer to room temperature (RT).

a. Mix by inversion to ensure all salts are into solution.

b. Prepare 1x assay buffer — dilute 1 part 10x assay buffer with

9 parts of dH20.

Sig 121210028259 Rev B

Bio-Plex Pro Assay Quick Guide Bio-Plex Pro Assay Quick Guide

B. Dilution of Standard (1:3 Serial Dilution)

1. Label 8 polypropylene tubes S1 through S8.

2. Transfer the reconstituted standard into the tube labeled “S1.”

3. Add the appropriate amount of the standard diluent into the labeled tubes according to the table below (this will be sufﬁcient for duplicate standard curves and blanks).

Standard Volume of Standard Diluent, µl Volume of Standard, µl

S2 100 50 of S1

S3 100 50 of S2

S4 100 50 of S3

S5 100 50 of S4

S6 100 50 of S5

S7 100 50 of S6

S8 100 50 of S7 Blank 100 —

4. Prepare working standards (S2–S8) by serial dilution. Transfer the appropriate volume of standard into each of the labeled tubes with standard diluent as outlined above.

5. Vortex each standard at a medium setting before proceeding with the next serial dilution. Change pipet tip at each dilution step.

C. Sample Preparation

1. Centrifuge samples at 500 x g for 5 min to remove particulates from all samples prior to use.

2. Prepare sample dilutions in 0.5 ml or 1.0 ml polypropylene tubes as required for the assay.

3. Dilution scenarios provided below are sufﬁcient to run each sample in duplicate.

Volume of Volume of Panel Sample Dilution Urine Sample, µl Sample Buffer, µl

Human Tox 1 1:4 20 60

Human Tox 2 1:50 10 490

Rat Tox 1 1:2 40 40

Rat Tox 2 1:50 10 490

Rat Albumin 1:10,000 5 (A. Prepare 1:100) 495 5 (B. Prepare 1:100) 495

Canine Tox 1 1:15 10 140

Canine Albumin 1:10,000 5 (C. Prepare 1:100) 495 5 (D. Prepare 1:100) 495

Note: Controls are ready to use after reconstitution. No dilution is needed.

D. Dispensing of Reagents

1. Add 10 µl of blocker to all wells of the plate.

2. Add 30 µl of the standard, control, sample, or blank to the appropriate well

of the plate.

3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of the

beads to all wells of the plate.

4. Cover plate with plate seal and protect from light with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.

5. Wash the plate three times with 100 µl 1x assay buffer.

6. Vortex the reconstituted detection antibodies at medium speed for 10–20 sec. Add 40 µl to each well.

7. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do not

aspirate after incubation.

8. Prepare the required dilution of SA-PE as outlined in the following table.

Note: Volumes in the table are for an entire 96-well plate. Smaller volumes

can be prepared, provided that dilution ratios are maintained.

9. Add 20 µl of diluted SA-PE to the required plate wells.