Dart Lab’s Millipore Luminex Record Sheet
For the Millipore protocol go to www.millipore.com. Type in your kit number and go to the protocol page.
Analytes: Custom 10-plex
IL-1ra, IL-1a, IL-6, IL-8, IL-10, MCP-1, MIP-1a, MIP-1b, Rantes, TNFa
Supplier: Millipore
Principal Investigator:
1 Plate
Type of Samples:
Plate: 1 of 1
Date:
# sample wells on Plate 1: 63
Total # wells on Plate 1: 96. Standards=18; Samples= 63; Blanks=3; Controls=12
Standards Plate 1: Use ______ as medium
Samples this plate:
Kit Used:
Medium:
Calibration: Broad
Standard Cat #:
Detection Antibody Catalog #:
Expiration:
Expiration:
Millipore Quality Control 1 Lot #:
Millipore Quality Control 2 Lot #:
Preparation of Plate
Prewet the filter plate by pipetting 200 l of Wash Buffer into each well of the Luminex Plate. Seal and shake
on a plate shaker for 10 minutes at room temperature.
Preparation of Beads
Sonicate the antibody bead vial for 30 sec; vortex for 1 min.
Volume to be added to wells: 25 l
For this 10-plex, there are 10vials/kit = 10 vials. Take 60 l from each vial. 0.060 ml x 10vials = 0.6 ml
beads. Bring this up to 3ml (for 1 plate) by adding 2.4 ml Bead Diluent for Hu kit, Assay Buffer for Mo kit.
# wells
96
Beads
0.6 ml
Diluent
2.4 ml
Total Bead Volume
3.0 ml
Bead count: Bead Count per 400 small squares:
Side 1: ______ Side 2: ______
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 (l)
1 sq. mm (400 small squares) x 0.1 mm (depth) x Number of Cytokines
i.e. Mean of duplicate bead count x volume beads added per well (l)
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 added 25 l beads per
well:
Mean = 54
54 x 25
= 3375 beads/cytokine/well
0.1 x 4
Calculation:
______ x 25
=
beads/cytokine/well
0.1 x 10
QA/QC: Beads/cytokine/well must be greater than 2000 to continue.
Dart Lab
Jacqueline Smith
16-Feb-16
PI
Date of Expt.
Kathy Smith
Page 1
Dart Lab’s Millipore Luminex Record Sheet
Preparation of Standard Curve
Diluent used: Deionized water
Dilution factor: 1:5
This is the most important part of the procedure. Be really accurate with volumes, i.e., no bubbles in tip.
Reconstitute lyophilised standard 1 with 250 l deionized water resulting in 10,000 pg/ml for all 10 analytes.
Invert vial 2-3x to ensure complete reconstitution. Vortex the vial for 10 seconds. Allow the vial to sit for 5 10 min.
Transfer the standard to an appropriately-labeled microfuge tube.
Vortex gently. This master mix will serve as 10,000 pg/ml Standard I in the Low PMT setting standard curve.
Standard 1 = 250 l of reconstituted lyophilised standard
Standard 2 = 50 l of standard 1 + 200 l of Assay Buffer
Standard 3 = 50 l of standard 2 + 200 l of Assay Buffer
Standard 4 = 50 l of standard 3 + 200 l of Assay Buffer
Standard 5 = 50 l of standard 4 + 200 l of Assay Buffer
Standard 6 = 50 l of standard 5 + 200 l of Assay Buffer
Vol of Blank to add to wells: 25 l;
Time: ____
Blank = 25 l of Assay Buffer in 3 wells.
Preparation of Controls
Use Quality Control 1 and 2 provided in kit as Controls, as well as Dart Lab Control at Neat and 1:100.
Make enough for 3 wells on 1 plate:
To make a 1/100 dilution and add 25 l to 3 wells: Diluent: Assay Buffer.
1/100: Of the approved hu DC exp’t aliquot, take 10 l spike + 990 l Assay Buffer = 1.0 ml 1/100 spike.
Aliquot 25 l in 3 wells on 1 plate.
Dilutions: Neat X
Low 1/100
X.
Preparation of Samples
Vol of Samples to add to wells: 25 l of samples
Are any samples diluted? No. Indicate on BioPlex software protocol.
Incubation time of beads and samples (1 hr for Hu; 2 hrs for Mo):
From ______________ to __________________
Preparation of Biotinylated Detection Antibody
Diluent: None-premixed in kit 3.2 ml in kit. Only need 2.64 ml
Multiply # of wells by 10% for spillage, etc.
# wells
96x10%
Amount to add: 25 l /well
Detection Antibody
2.4 l x 1.1% = 2.64 ml
Incubation time (0.5hr for Hu; 1 hr for Mo): From ______________ to __________________
******DO NOT Vacuum Det Ab after incubation. Add Strep-PE directly to Det Ab. *********
Dart Lab
Jacqueline Smith
16-Feb-16
PI
Date of Expt.
Kathy Smith
Page 2
Dart Lab’s Millipore Luminex Record Sheet
Preparation of Streptavidin-PE Working Solution:
Diluent: None-premixed in kit
Amount to add: 25 l /well
DO NOT Vacuum Det Ab after incubation. Add Strep-PE directly to Det Ab.
# wells
Streptavidin-PE
96x10%=106 wells
2.4 ml x 10% = 2.64 ml
Incubation time (30 min for either Hu or Mo): From ______________ to __________________
Calibration of Bio-Plex Reader:
Broad
X
Time Plate 1 Read Start: _________ Finish: ________
Remove stickers from vials and tape here
Catalog #
Lot #
Expiration date
Beads
Standards
Detection antibody
Spike
Streptavidin-PE
Dart Lab
Jacqueline Smith
16-Feb-16
PI
Date of Expt.
Kathy Smith
Page 3