Luminex Requisition Form
Lab PI ______Phone #______Lab Phone #______
Person requesting Luminex ______
Today’s Date ______Scheduled Date of Assay ______
Account string to be charged______
Cytokine Kits
Mouse 32-plex (Millipore)
Samples
Type: Delivered:
_____ Cell culture supernatant _____ in U-well plate
_____ Serum
_____ Plasma
_____ Tissue homogenate
_____ Lavage fluid
_____ Other (describe) ______
Sample medium (except serum and plasma samples):
What medium are your samples in?
___ RPMI ___ AIM V ___ PBS Other (describe)______
Does your medium contain FBS, FCS, HS, or BSA? _____ NO _____ YES
Which? ______How much?______%
Cytokine concentrations:
The standard curve for each cytokine ranges from ~2 - 32,000 pg/ml.
Number of samples: Dilution factor:
_____ singles _____ undiluted
_____ duplicates _____ ?-fold
_____ triplicates _____ pre-diluted in your lab
*_____ Total # of wells _____ Dart Lab to dilute (Charge $)
Are any or all of your samples HIV/AIDS positive? ______
Hep B positive? ______
Hep C positive? ______
If so, on the attached plate map, please indicate which samples are positive and for which disease.
Sample Amount: Please deliver in U-well plate according to provided plate map
Millipore kit: 40ul supernatant (see kit protocol for plasma or serum dilution) samples
Sample Identification: Please send an Excel file to: ajb@dartmouth.edu and r
with your sample identification information (i.e., Pt 456 Mo. 2) in one Excel cell/sample.
Luminex plate format:
Capacity of each kit is 66 wells of samples, e.g. 22 triplicates:
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12A / Standard / Standard / Standard / QC 1 / 3 / 11 / 19 / 27 / 35 / 43 / 51 / 59
B / Standard / Standard / Standard / QC 1 / 4 / 12 / 20 / 28 / 36 / 44 / 52 / 60
C / Standard / Standard / Standard / QC 1 / 5 / 13 / 21 / 29 / 37 / 45 / 53 / 61
D / Standard / Standard / Standard / QC 2 / 6 / 14 / 22 / 30 / 38 / 46 / 54 / 62
E / Standard / Standard / Standard / QC 2 / 7 / 15 / 23 / 31 / 39 / 47 / 55 / 63
F / Standard / Standard / Standard / QC 2 / 8 / 16 / 24 / 32 / 40 / 48 / 56 / 64
G / Standard / Standard / Standard / 1 / 9 / 17 / 25 / 33 / 41 / 49 / 57 / 65
H / Blank / Blank / Blank / 2 / 10 / 18 / 26 / 34 / 42 / 50 / 58 / 66
Points to consider BEFORE harvesting samples:
● We need to dilute the cytokine standards in the identical medium your samples are in. So freeze 10 ml culture medium (e.g. RPMI/10% FBS) at experiment set-up.
● For whole blood, collect plasma rather than serum. The clotting process causes platelet degranulation. Platelet granules are a source of many cytokines.
● Harvest your samples then centrifuge them 14,000 rpm for 3 minutes in a cold microcentrifuge (to pellet particulates). Remove supernatants, avoiding any lipid layer, and aliquot.
● Do not freeze and thaw samples more than once. Cytokines deteriorate with freezing and thawing. Aliquot and freeze small volumes (~180 ul for triplicates) at harvest.
● If serum-free culture medium is used, add 0.5% BSA (5 mg/ml) as carrier protein before freezing.
Points to consider when preparing samples to be given to Dart Lab:
Warning: Hemolyzed samples are not suitable for Bio-Plex cytokine assays and will not be accepted (the instrument gets clogged).
Thaw samples the morning of the assay. Samples must be brought to DartLab before 10am the morning of the assay.
Keep all thawed samples on ice until ready for use.
We add 25uL sample per well, so provide us with at least 40uL to ensure adequate pipetting.
Add your samples to a U-bottom 96-well plate as shown in the Luminex template.
If your samples need to be diluted, follow the recommendations in the Table:
Serum Isolation
Allow the whole blood samples to clot for 1–2 hr at 37°C. Alternatively, use a serum separator tube and allow the blood samples to clot for 30 min. Centrifuge at 1,000 x g at 4°C. Collect the serum, avoiding any lipid layer, and assay immediately or freeze at –20°C in small aliquots.
Plasma Isolation
Sodium citrate tubes are recommended; EDTA tubes are acceptable, but sodium citrate yields less clumping. Centrifuge at 1,000 x g at 4°C for 10 min. Collect the supernatant and either filter through a sterile 0.22 μm filter or centrifuge 14,000 rpm for 3 minutes in a refrigerated microfuge. Collect the plasma, avoiding any lipid layer, and assay immediately or freeze at –20°C.
Dart Lab 23-Jan-15