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 1 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 Kathy.Smith@dartmouth.edu 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 20-Dec-10 1