Dart Lab’s Bio-Rad Luminex Record Sheet
For Bio-Rad’s protocol, go to www.Bio-Rad.com.
Analytes: Human 6-plex
IL-2, IL-5, IL-9, IL-10, IL-13, VEGFa
Supplier: Bio-Rad
I mixed 6 vials of beads & det ab
PI:
Magnetic kit
1 Plate: 96 wells (63 sample wells)
Plate: Pl 1 of 1
Type of Samples:
Date:
# sample wells on this plate: 63
Total # wells on this plate: 96. Standards=24 Samples=63; Blanks=3; Controls=6
Standards Diluent:
Samples this plate:
Pl 1: 96 (24 standards; 63 samples; 3 blanks, 2 sets of control in triplicates (1/3, 1/400 in
Assay buffer)
Kit Used:
Medium:
Calibration: BROAD
kms
Preparation of 10X Beads: I mixed 6 vials of beads
Diluent: Assay Buffer A
Volume to be added to wells: __50___ l
# wells
96
10x Beads (36ul)
575x6=3450 ul
Assay buffer (24ul)
2.3 ml
Bead count:
Bead Count per 400 small squares:
Side 1: ______ Side 2: ______
Total Volume (60ul)
5.75 ml
Mean: ______
Use the following formula to calculate if there are sufficient beads for the assay:
Mean of duplicate bead count x 1000 (mm3 to cc3 conversion factor) x volume beads
added per well (ul)
1 sq. mm (400 small squares) x 0.1 mm (depth) x Number of Cytokines x
1000
i.e. Mean of duplicate bead count x volume beads added per well (ul)
0.1 x number of cytokines
= beads/cytokine/well
Example: if doing a 4-plex, have counted 56 and 52 beads in 400 small squares, and
1
Dart Lab
PI
Date of Expt
Jacqueline Smith
Nov. 11, 10
Kathy Smith
Dart Lab’s Bio-Rad Luminex Record Sheet
added 50 ul beads per well:
Mean = 54
Calculation:
54 x 50
= 6750 beads/cytokine/well
0.1 x 4
______ x _50___
=
beads/cytokine/well
0.1 x 6
QA/QC: Beads/cytokine/well must be greater than 2000 to continue.
Preparation of Standard Curve
Made 1 set of standards.
Diluent used: ________
This is the most important part of the procedure. Be really accurate with volumes, i.e., no
bubbles in tip.
1. Lyophilised standard 1 Gp I reconstituted with _500_ ul _(diluent)_______, resulting in
22147 pg/ml IL-2.
Vortex gently 1-3 sec and incubate on ice for 30 min. Equilibrate 20 min. at RT.
Vortex gently. This master mix will serve as Standard 1 in the Broad PMT setting standard
curve.
Standard 1 = _256_ l of stock
+ ____ __144____ of diluent.
Time: ___________
Equilibrate:_______
Standard 2 = _100_ l of standard 1 + _____300____ of diluent. Start dilutions: ____
Standard 3 = _100_
l of standard 2
+ _____300____ of diluent.
Standard 4 = 100_
l of standard 3
+ _____ 300____ of diluent.
Standard 5 = _100_
l of standard 4
+ _____300____ of diluent.
Standard 6 = _100_
l of standard 5
+ _____300____ of diluent.
Standard 7 = _100_
l of standard 6
+ _____300____ of diluent.
Standard 8 = _100_ l of standard 7 + _____300____ of diluent.
Vol of Standard to add to wells: __50ul__
I ran 1 set of Blanks: 3 wells @ 50 l/well of_____.
Preparation of Controls (Spikes) Add 50 ul to each well. Diluent: Assay Buffer.
QA/QC: A high and low spike should be also be added to each plate as an internal Quality Control. As of 20 Apr
06 Dart Lab has used pooled supernants from Human DC experiments as Biological Controls.
1
Dart Lab
PI
Date of Expt
Jacqueline Smith
Nov. 11, 10
Kathy Smith
Dart Lab’s Bio-Rad Luminex Record Sheet
Make enough Control for 1 plate: To make a 1/3 and 1/400 dilution and add 50ul to 6 wells:
Diluent: Assay Buffer.
1/3: Of the approved Hu DC/PBMC exp’t aliquot, take 100 ul + 200 ul Assay Buffer = 300ul
1/3 spike. Plate 50ul in triplicate. Use 10ul for 1/400 spike.
1/400: 1/3 x 1/133: Take 10 ul 1/3 spike + 1.32 ul Assay Buffer = 1.33 ul 1/400 spike. Plate 50
ul in triplicate.
Dilutions: High 1/3 __X__ Low 1/400 ___X___ .
Vol of Spike to add to wells: _____50____
l.
Preparation of Samples
Vol of Samples to add to wells: _50__
l.
Are any samples diluted? _No.__ Indicate on BioPlex software protocol.
Incubation time of beads and samples (30 min):
Pl 1 From ______________ to __________________
Preparation of Biotinylated Detection Antibody (DA):
Diluent: Detection Antibody Diluent A
Amount to add: 25 ul/well
# wells
96
10x Det Ab (18.75ul)
300x6=1800ul
Det Ab Diluent (12.5ul)
1.2 ml
Total Vol (31.25ul)
3.0 ml
Incubation time of detection antibody (30 min):
Pl 1 From ______________ to _______________
Preparation of Streptavidin-PE Working Solution:
Diluent: Assay Buffer
Amount to add: 50 ul/well
# wells
96
100x Strep-PE (0.625ul)
60ul
Assay Buffer (62ul)
5.94 ml
Total Vol (62.5ul)
6.0 ml
Incubation time of Streptavidin-PE (10 min):
1
Dart Lab
PI
Date of Expt
Jacqueline Smith
Nov. 11, 10
Kathy Smith
Dart Lab’s Bio-Rad Luminex Record Sheet
Pl 1 From ______________ to __________________
Calibration of Bio-Plex Reader:
Broad ___X____
Time Plate 1 Read Start: _________ Finish: ________
Remove stickers from vials and tape here:
catalog number lot number
expiration dates
Beads
Standards
Detection
antibody
Streptavidin-PE
Spike
1
Dart Lab
PI
Date of Expt
Jacqueline Smith
Nov. 11, 10
Kathy Smith