Luminex xPONENT 4.0 SP1 - Manual (Page 17)

5
Introduction
measured and used to determine the result of each assay in a multiplex. Again, since
each bead is analyzed individually, reporter signals for each bead set can be accurately
quantified.
The following table displays acceptable reporter fluorochromes and their excitation and
emission wavelengths.
Sample Volume
Sample volumes or sample sizes range from 10 to 200 µL. Ensure that some sample
remains in the well after aspiration; about 25 µL greater than the sample volume. This
amount may vary depending on the type of plate used. After acquisition, the Luminex
analyzer washes the sample lines resulting in ejection of approximately 150 µL of sheath
fluid back into the well for a 96-well plate. Ensure that there is room to add this amount to
the well without overflowing and contaminating other wells.
The volume restrictions on the assay design can be expounded by the following formula:
Total well volume (µL) – Sample uptake volume (µL) + 150 (µL) (µL)
• Total well volume = Starting sample volume of a well before the unit samples for
acquisition. Well volume is determined by the consistency of the bead set.
• Sample uptake volume = Uptake volume for acquisition (program this in the protocol as
sample volume).
• 150 (µL) = Volume expelled back as stated in the above paragraph.
• Maximum well volume plate = The maximum volume capacity of the wells in a selected
96-well microtiter plate.
NOTE: This sample volume information is for a 96-well plate. For
information regarding 384-well plates, refer to the assay kit
instructions or contact Luminex technical support.
Sample Dilution
Dilute concentrated biological samples, such as plasma or serum, at least 1:5 with
reagents as part of assay setup or as a final dilution step. If running an xMAP-based kit,
follow the dilution instructions found on the kit’s product insert.
TABLE 2. Reporter Fluorochromes Wavelengths
R-Phycoerythrin Alexa 532
Formula weight (Daltons) 240,000 470
Absorbance max (nm) 480, 546, 565 531
Extinction max (M-1cm-1) 1,960,000 83,800
Emission max (nm) 578 554
Quantum yield 0.82 0.8