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Luminometers with and without injectors


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#1 cell_man

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Posted 22 October 2009 - 08:49 AM

Hi,

I am trying to transfect luciferase expressing plasmids in to 293 cell line and determine the flourescence intensity by using a luminometer with an injector. For this type of luminometer, the assay solution should be injected in to the system before measuring the intensity. We recently received this machine from a lab and no one around knows how to inject the solution. My 1st question obviously is how do you guys inject the solution (if you have a photographic reference, it would be great). My 2nd question is can I use the protocol for luminometer without the injector for this machine? my 3rd question is what difference does the injector makes?

THanks a lot in advance.

#2 96well

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Posted 02 December 2009 - 02:16 PM

Hi,

I am trying to transfect luciferase expressing plasmids in to 293 cell line and determine the flourescence intensity by using a luminometer with an injector. For this type of luminometer, the assay solution should be injected in to the system before measuring the intensity. We recently received this machine from a lab and no one around knows how to inject the solution. My 1st question obviously is how do you guys inject the solution (if you have a photographic reference, it would be great). My 2nd question is can I use the protocol for luminometer without the injector for this machine? my 3rd question is what difference does the injector makes?

THanks a lot in advance.


Hi,
standard injection settings for luciferase assays are:
your cell lysate = 20 uL,
luciferin injection volume: 100 uL,
integration time: 8-10 seconds.

before injecting, obviously you need to charge the injector pump with luciferin (aka, fill the plastic tubes), this procedure is usually named as "priming". It's a critical step: some air bubbles enter in the line (you see them), but with consecutive priming steps they exit: some air while injecting luciferin in the plate can result in not accurate volume injection to null injection.

manuals for luminometers are usually free available online with photographic references at the site of luminometer manufacturer.

Injectors were adopted to allow the measurement immediately after the injection of luciferin (fractions of second after the injection). This should allow to capture the initial light-emission of luciferase (there is a peak of light at the beginning which is brighter than the following steady state). Thus, using injector would allow more sensitivity. However, the luminometer can also read the luminescence without automatic injectionan. This is useful for other assas like luminescent HRP-based ELISA.

Finally, injectors increase the cost of the luminometer to 1000-to-3000$.

more info about luciferases on my blog: www.reportergene.com

keep great reporter assay!
Nature magazine. Do you qualify for a free subscription?

#3 NWU99

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Posted 03 December 2009 - 11:05 AM

Several factors make injectors useful for luciferase assays:

-For luciferase, if you inject inside the reader, you don't miss interesting fast reaction events that would be missed otherwise. This is also important for calcium measurements using aequorin, enzyme kinetics and ATP detection. The LUMIstar and FLUOstar Omega plate readers inject at the point of measurement, so you don't miss any initial reactions fast kinetic..
-For low-sensitivity luminescence, reading immediately after injection allows you to measure when the light emission is most intense.
-Using dispensers allows you to measure at the exact same time relative to injection for every sample, improving assay consistency.
-Injectors allow you to read more samples faster and it's also easier to make adjustments, compared to a manual pipettor.

I work for BMG Labtech, so please get in touch if you have any specific questions. Good luck!




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