Frequently Asked Question's:

Q. What kind of test tubes should I use with my Optocomp luminometer?

A: Polystyrene, polypropylene and glass test tubes can all be used, however there are some important considerations. Polystyrene test tubes can accumulate static electricity, which can cause erratic high backgrounds when it discharges. This effect can be minimized by carefully wiping the outside of each test tube with a clean lint-free wipe (such as a Kim-Wipe) lightly moistened with water prior to inserting it into the instrument. This removes the static charge from the exterior of the test tube. Glass tubes will not suffer from static electricity, however glass tubes can phosphoresce after exposure to fluorescent light or sunlight (any light containing ultraviolet light). The phosphorescence can also cause elevated background. If you are considering glass tubes, be sure to select tubes made from borosilicate glass. Borosilicate glass usually has lower levels of the contaminants that phosphoresce, however, some borosilicate glass test tubes will phosphoresce. You can check for phosphorescence using the method described in Tip 4, below. Polypropylene tubes usually do not suffer from either static electricity nor phosphorescence.

Q. What is the minimum sample volume I can use in an Optocomp?

A: For the Optocomp I and II; anywhere from virtually no sample up to about 1.5 ml (1,500 microliters) will work well. Above about 1.5ml part of the sample will not be directly exposed to the PMT, however this may not affect your results, as long as all of your samples are the same volume and are reasonably homogeneous. Sample volumes above 1.5ml are not an issue if you are not attempting to quantify the analyte. For the Optocomp P the range would be from virtually no sample up to about 1.1ml (1,100 microliters).

Q. Are there any reagents that are not compatible with the Optocomp's injectors?

A: The fluid path in the Optocomp's injectors is constructed entirely of Teflon and PEEK, two extremely inert materials. The injector system is compatible with all of the popular reagent systems and chemiluminescent and bioluminescent assay kits.

Q. What is an RLU?

A: RLU stands for "Relative Light Unit". This term is used because luminometers typically do not yield a measurement directly in units of photons. This is because some luminometers measure the current output by the photomultiplier tube, which cannot be translated into photons, while in the case of photon-counting instruments, the effective bandwidth of the counters used to accumulate the photon count is usually extended by dividing (pre-scaling) the signal from the photomultiplier tube by some constant (usually 10) using a high-speed counter. Additionally, a calibration factor is typically used to normalize the response of the instrument to a calibrated light standard.

Q: My Optocomp I (or Optocomp P) does not have "Dual Read" software. How can I best run Promega's Dual-Luciferase Reporter assay (DLR assay) on my Optocomp I?

A: The Dual-Luciferase Reporter assay is actually very flexible, and can be run with a variety of luminometer set-ups. The DLR assay can be run using an Optocomp I with or without injectors, or even on an Optocomp P, which is not available with injectors. If you have two injectors, you can have the instrument add both the Luciferase Assay Reagent II (or LAR II, which initiates Firefly Luciferase luminescence) and the Stop & Glo reagent (which quenches the Firefly Luciferase luminescence and initiates Renilla Luciferase luminescence) to your samples. If you have only a single injector, then you can either use the injector to add one of the reagents and manually (pipette) add the other, or you can change reagents used in the injector in the middle of the assay. In either case (one or two injectors) the injector(s) should both be set to deliver 100 microliters of the reagent. Or, you can use an Optocomp without injectors, adding both reagents manually to your samples.

In any case, you should program two protocols for the assay. One protocol will measure Firefly Luciferase in your samples and the other will measure Renilla Luciferase. Simply start the first protocol, and run all of your samples using the LAR II reagent. Then start the second protocol and run all of your samples again, this time using the Stop & Glo reagent. The delay between addition of the LAR II reagent and the Stop & Glo reagent will not affect your results. Of course, if you are using injector(s) to add both reagents, then you need to have the appropriate injector primed with the proper reagent before starting each protocol.

Dual-Luciferase and Stop & Glo are trademarks of Promega Corporation

Q. How can I determine how light emission from my samples changes over time?

A: With the Optocomp I and the Optocomp II, you can use the Kinetic protocol. You can program a Kinetic protocol to divide each measurement into up to 100 time slices, or intervals, with each interval programmable from 20 milliseconds up to 650 seconds. After each sample is read, the Optocomp will print the total RLU measured as well as the RLU for the peak interval. You can then choose to plot a histogram of the light emission, and/or print the data for all of the intervals.

Q: What kind of standard can be used to calibrate my Optocomp luminometer?

A: A standard is available from MGM Instruments for this purpose. The standard contains a very small amount (less than 10 microcuries) of Tritium. Tritium has a half-life of about 12 years, so the activity of the standard must be corrected for decay of the Tritium. A decay chart is provided with each standard for this purpose.

Q: What kind of standard can be used to calibrate my Isocomp gamma counter?

A: A standard is available from MGM Instruments for this purpose. The standard contains a very small amount (less than a tenth of a microcurie) of I129, which is used to simulate the I125 typically used in many radioisotope-labeled assays. The energy levels of the x-ray and gamma radiation of the two isotopes are very similar, making use of I129 practical. I125's half-life (60 days) makes its use as a standard impossible, while I129's half-life of 15.7 million years makes it a stable source suitable for use as a calibrator.

It is extremely important that the Isocomp is properly calibrated before running an assay. The Isocomp accepts a narrow range of radioactivity based on its energy, this is done to exclude background radiation from your measurements. As a result, if the instrument is not properly calibrated, it is quite possible that little or none of the actual signal might be measured, causing serious error in the assay.

Tips

1. Tip: To insure accurate results, you should calibrate your Isocomp every day, before running assays!

2. Tip: To insure reliable performance from your Optocomp's injectors, be sure to flush them out thoroughly immediately after you are done using the instrument. Many reagents used with luminometers will clog the fluid path if left in the injector. You can flush your injectors out with DI water. If they have become clogged try flushing them with warm DI water or a mixture of DI water and methanol.

3. Tip: If you suspect your Optocomp I or Optocomp P has background problems, try making a measurement without a sample tube in the instrument. This will allow you to determine exactly what the instrument's background is. This is important, because we find that usually the problem is with the samples or the test tubes, and not the instrument.

For the Optocomp I, first, start a protocol that does not have injectors programmed in it (the count time should be set to one second). Next, notice that there are two round black plastic devices in the sides of the recess the lid closes into, to either side of the sample position. One of these is an infrared emitter and the other is a detector, these are used to detect the presence or absence of the sample test tube in the measurement position. With the lid completely open, place your finger over one (it doesn't matter which) of these little round black plastic rimmed holes. Now, holding your finger there, close the lid part way, then remove your finger and finish closing the lid. The Optocomp I will now be fooled into believing a sample tube is in the measurement position, and will proceed to acquire it. To make repeated measurements simply hold the lid closed.

To check the background in an Optocomp P portable luminometer simply close the turret without placing a sample tube into the turret. The Optocomp P does not have injector capability, and so does not require a means to detect whether a sample tube is present.

Unfortunately, instrument background cannot be easily checked in the Optocomp II.

4. Tip: To determine if your test tubes are phosphorescing, run a raw data protocol programmed for a one second count time and no injectors. After determining the instrument background using the method described in the previous tip, hold each of the test tubes you wish to check, empty, relatively close (two or three feet away is fine) to a fluorescent light for several seconds, then immediately and quickly place each tube into the instrument and close the lid (in an Optocomp I), or rotate the turret closed (in an Optocomp P). Hold the lid down and allow the instrument to measure the test tube repeatedly across a period of twenty or thirty seconds. Any phosphorescence will be seen as an elevated background reading that will gradually decline over the period of the measurement until normal instrument background is reached.

It is difficult to measure phosphorescence of test tubes in an Optocomp II, because of the time it takes to place the tube(s) into the cassette(s) close the lid and initiate the measurement. Fortunately, because you typically load some number of tubes into an Optocomp II and then start the protocol, any phosphorescence will usually have decayed before the tubes are read. If you suspect your measurements are being affected by phosphorescence of the test tubes, then simply load your samples into the instrument, close the lid, and wait a minute or so before starting the protocol.