Bio-Rad Rat Diabetes Assays - Manual (Page 16)

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Please skip this section if not mixing diabetes with cytokine assays.
Reconstituting Standards for Cross-Panel Plexing
Follow these directions when mixing human or mouse diabetes and
cytokine assays. Note that rat diabetes and cytokine standards are
premixed into one standard vial. Therefore, no extra mixing is required.
Two mixing scenarios are provided in Figures 4 and 5 for detection at high
and low PMT respectively. One results in a narrow range cytokine standard
curve for detection at high RP1 (PMT); the other gives a broad range
cytokine standard curve for detection at low RP1 (PMT) setting.
1. Gently tap both vials of lyophilized diabetes and cytokine standards.
2. For high PMT setting/narrow range cytokine standard curve, add
500 μl of the appropriate standard diluent to each vial. For low PMT
setting/broad range cytokine standard curve, add 250 µl of diluent to
each vial. Do not use assay buffer or sample diluent to reconstitute
the standards.
3. Gently vortex the reconstituted standards for 5 sec then incubate
on ice for 30 min. Be consistent with the incubation time in every
assay to ensure best results.
4. During the incubation period, prepare the samples as instructed in the
Prepare Samples step.
Preparing Serial Dilutions
Pipet carefully with calibrated pipets, and use new pipet tips for every
volume transfer.
1. Label nine 1.5 ml polypropylene tubes S1 through S8 and Blank.
2. For high PMT/narrow range cytokine standard curve, add 59.2 µl of
standard diluent to the S1 tube. For low PMT/broad range cytokine
standard curve, add 72 µl of standard diluent to S1 (Figures 4 and 5).