Download Rat Supplier (check one): ____ Bio

Luminex Requisition Form
Lab PI ______________ Phone #_____________
Lab Phone #_____________
Person requesting Luminex _____________________________________________
Today’s Date ________________
Scheduled Date of Assay ______________
Account string to be charged_____________________________________________
Luminex kit order to be placed by the Immune Monitoring Laboratory
Species
_____ Human
_____ Mouse
_____ Rat
Supplier (check one): ____ Bio-Rad
____ Millipore
Cytokine Kits
____ Human 10-plex high sensitivity (Millipore)
____ Human 23-plex Gp II (Bio-Rad)
____ Human 27-plex Gp I (Bio-Rad)
____ Human 42-plex (Millipore)
____ Mouse 9-plex Gp II (Bio-Rad)
____ Mouse 23-plex Gp I (Bio-Rad)
____ Mouse 32-plex (Millipore)
____ Custom cytokines of your choice, e.g. Mouse or Human 2-plex (IL-2, IL-4)
(go to Dart Lab Website http://www.dartmouth.edu/~dartlab and check the Link to
‘Luminex Cytokine Kits’)________________________________________________
Samples
Type:
Delivered:
_____ Cell culture supernatant
_____ in U-well plate
_____ Serum
_____ in individual vials
_____ Plasma
_____ Thawed day of run
_____ Tissue homogenate
_____ Thawed day before run
_____ 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? __________ %
Please provide 10 ml of your medium when you bring your samples.
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
*Required:_____ Total # of wells
_____ request Dart Lab to dilute
Are any or all of your samples HIV/AIDS positive? ___________
Hep B positive?
___________
Hep C positive?
___________
Dart Lab
20-Dec-10
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Luminex Requisition Form
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 acc to provided plate map:
Bio-Rad kit: 60ul of supernatant or 15ul of serum/plasma samples;
Millipore kit: 35ul supernatant (see kit protocol for plasma or serum dilution)
samples
Sample Identification: Please send an Excel file to [email protected]
with your sample identification information (i.e., Pt 456 Mo. 2) in one Excel
cell/sample.
Multiple Diluents: A separate set of standards (preferably run in triplicate) is
required for each cell diluent. Please keep in mind, if more than one type of
diluent is used, this greatly reduces the available number of wells for samples.
Luminex plate format:
Capacity of each kit is 63 wells of samples, e.g. 21 triplicates:
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. 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.
Keep all thawed samples on ice until ready for use.
We add 50 ul sample per well, so provide us with at least 60 ul.
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:
Cell culture
supernatant
Dart Lab
20-Dec-10
Serum-free cell Serum/plasma
culture
supernatant
1
Lavage samples
Luminex Requisition Form
Freezing
Diluent
add 0.5% BSA
culture medium
Volume to add 60 ul
per well
culture medium
60 ul
add 0.5% BSA if
serum-free
Bio-Rad: 1 part serum to 3 lavage buffer
parts Bio-Rad Serum
Diluent .
Millipore: see kit protocol
Bio-Rad:15 ul diluted 1:4
Millipore: see protocol
60 ul
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
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