Luminescent Measurements - Science topic

Hey all,

I am doing experiments on cultured cells and need to perform an apoptosis-assay. My supervisor recommended me the Caspase3/7 Glo Assay from Promega and told me to use this (briefly described) protocoll:

- cells were cultured in 6 well lates and lysates in protein lysis Buffer (containing EDTA and phosphatase/proteinase inhibitors) and zentrifuged

-equal amounts of (fresh) cell lysate and Caspase 3/7 reagent would be pipetted in a 96 well plate and incubated for 1-2h

-measure luminescence

As the protocoll provided by the manufacturer is quite diffrent than this (culture cells directly in 96 well plate, measure in cell culture media (living cells) and not in lysate) I wanted to ask if anybody perform this assay as described here or can even recommend me another way to adopt the protocol as I am quite sceptical about this.

Another question would be, if anybody used stored cell lysates (-20 degrees) for this assay as well, as this question arised as well in my discussion with my supervisor. I highly doubt this, but I wanted to ask anyway.

Thank you for your help in advance!

What is the meaning of diffuse and specular reflectance in the case of UV VIS when we are recording in the form of powders (glasses)?

Currently doing research on mechanoluminescence materials, and I plan to create a measurement set-up. The aim is to measure luminescence (wavelength and intensity) properties while the material is compressed by a universal testing machine. Is there anyone who's familiar with this? If so, I might also need recommendations on measurement tools (spectrometer etc.) that I should buy for measurement.

Aside from the wavelength and intensity, is there any properties that is important for mechanoluminescence materials?

I decided to measure luminescence of firefly luciferase in Arabidopsis leaf discs using a Corning 96-well white opaque plate and a CCD (PIXIS 1300B). I chose the white opaque plate to minimize neighbouring well crosstalk.

When taking initial images, areas outside the wells are brighter than the wells themselves, what is causing this increase in background? Could there be fluorescence after exposure to the ambient room light? Why might this be specific to the centre of the plate?

The bottom two rows are blank.

I ended up using a plate reader to measure luminescence, but was initially curious what it would look like underneath a CCD.

Hi all,

I have generated two gene expression curves by measuring bioluminescence every 10 mins for 12 hrs. These curves tend to overlap at at some time points, while a difference can be seen at other time points. What is the best way to statistically analyze the curves and differences (if any)?

Thanks,

Hi,

I run my experiment with either red or green laser and I use the PPFD meter to measure the intensity before my experiment. Does it make any sense for monochromatic light? What would be the difference if I have 50 micro-mol/m².s for green laser and 50 micro-mol/m².s for red laser. Can I consider these two values equally intense or bright?

Hello,

I am using a lentivirus encoding CLuc to transduce target cells. I want to then lyse the cells in the presence of luciferin and measure luminescence. The kit which I had hoped to use is Steady-Glo (Promega). This reagent contains beetle luciferin. Will my CLuc catalyze the conversion of this beetle luciferin? Or do I need a kit which contains Cypridinid luciferin, AKA Vargulin?

Thank you!

I am using a QuickTiter Retrovirus Quantitation Kit and the protocol says to "read the microplate using a 480/520 nm filter set", but I am unsure as to what this means. Is this suggesting that there should be two excitation wavelengths, or that one wavelength is for background? I am currently using a SpecrtaMax L luminescence microplate reader with Softmax Pro software and I cannot determine the correct settings to use.

I need to know the best way to measure Human Chorionic Gonadotropin in culture supernatant and serum. Most of the literature use anelectro chemiluminescence assay. ELISA kits are available from RandD and in vitrogen although published studies utilising these kits are lacking. What has anyone used and if you've used the above methods how does the ECL compare with ELISA ?

Hello all,

I would like to know how do the covalency and asymmetry correlate with the measured luminescence lifetime values?

Thank you

Rim

should be necessary in such rigid matrix? solvent could be CH2Cl2

I have to do some bioluminescence assays, in that regard if anyone has a caspase-3 or any other caspase-luciferase construct, kindly let me know.

Hello everyone,

I have been using KRP as a media for PMNs during ROS assays (luminescence based detection of ROS generation upon infection of PMNs with Strep Pneumo).

I couldn't find in the literature why KRP is the buffer of choice for such experiment.

Does anyone knows why KRP is used instead of let's say HBSS-?

Thank you

Hello all,

I wish to perform a luciferase luminescent measurement of ATP in E. coli.

I have acquired a kit that requires less than 1 µM of KCl (and other monovalent metal salts). Also divalent metals must be in the ~3 mM range, and EDTA inhibits the reaction.

Normally, I would perform serial dilutions of the cells in PBS, but this raises the concentration of salts too much.

I thought about using Tris-HCl 0.01 M, but the only protocols i found are for cell lysis. I want to lyse the cells but not immediately.

Can i use Tris 0.01 M at 7.4 pH for the serial dilution of my cells without them suffering stress?

Tuning luminescence of platinum complexes is well established by variation of cyclometalating ligand. The most common trend I find in literature is a red shift in the emission upon introducing more electron rich rings to the ligand (for example replacing phenyl with thiophene). I am observing the exact opposite, in which more electron rich ligands will result in blue shifted emission and was wondering what might be the possible reason ?

How can I convert lux into PPF (μmol m-2 s-1) for Red and Blue LEDs light?

Emission starts from within the excitation volume in the material. Looking from the top, this volume can be much bigger than the spatial extent of the point in the CL map for which the intensity is recorded.

Hence, a bright point (pixel) in CL map does not mean that 'higher emission is generated from that particular point' but rather 'it is from the larger excitation volume associated with that smaller point'. Is it correct?

We have spurious signal from empty sphere. We use sphere covered with Spectralon ( G8 or Quanta Phi). It seems that this sphere was contaminated. But we never used it before and it is brand new. I would like to communicate with somebody who do such measurements personally. I have some questions regarding measurement procedure.

Currently i am working on alumina film coating for optical application. I have done my Photo luminescence measurement for deposited alumina film. and i have got some of the intense emission peak at 657nm and second at 824nm. can anyone help to distinguish these emission peaks?

I get vacuum UV photoluminescence specktums of rare earth doped inorganic compounds. when I check the literature to determine luminescence transitions, I came across emission spectras and excitation spectras. As an chemist, discrimination of these spectras is very hard study for me. Could someone explain this trouble?  

I am trying to measure change in cellular calcium ion concentration using aequorin expressing plant. I could find many research article and protocols about the measurement using the same system, so I summarized and followed them. However, the signal seems so weak even when I applied twice as much amount of coelenterazin as previous reports. It required at least 30s exposure for our instrument, therefore, not applicable for the purpose of my study. The 30 seconds exposure still does not show any distinctive difference between non-expressing samples and aequorin expressing samples. Even after discharge with CaCl2 and methanol, the signal doesn’t get any stronger. The instrument is NightShade, and sensitivity of the instrument seems fine, based on other luminescence measurement such as luciferase expressing sample treated with luciferin.

If you have an experience in this experiment, please let me know how to solve the problem.

Thanks in advance.

I working with Carbon nanodots(c-dots) I would like to measure the Quantum yield for prepared samples and also I am very eager to measure the Two Photon Absorption(TPA). In order to realize the above both measurements i need to know the concentration of C-dots in solutions as well as my C-dots are water soluble. How could I do this? I want the details of this. Thanking you in advance all researchers. 

I have faced similar problem in different spectrometers and hear a lot also about it from various researchers. Now I am wondering if any option can lit!

The Horiba DAS6 software in my computer is down. If you have the software in your lab, wound you mind helping me to fit the .das data into first order exponential decay and export the outcome to .txt file.

I attached my .das files in the attachement.

You help is very much appreciated!

Thanks!

On Belkin et al (1996) and later studies, kanamycin was omitted due to inhibit protein synthesis as it's mechanism of action but on van Dyk et al (1994) kanamycin was included in luminescence reading.

When ampicillin used instead of kanamycin, no luminescence inhibiton was observed. E. coli bioluminescence contain pUCD615 with luxCDABE, recA/grpE/fabA/katG promoter and kanamycin and ampicillin resistance.

I have added Ag nano particles in Photo luminescent dye solution in order to increase its florescence emission intensity. But unexpectedly its intensity start decreasing. I have use 15ppm to more then 200ppm for finding optimum value. but in all cases intensity decreases. but according to literature, it should increase. can anybody guide me please.. I thought i may be using high concentration which causes aggregation quenching effect but when use low concentrations, results were still opposite to literature.

I am attempting to obtain the fluorescence emission spectra from nanocrystals that I have prepared.  The spectrophotometric equipment utilizes a fiber optic cable (1 meter long) to collect the emitted light and bring it to the spectrophotometer.  This fiber optic cable is of the UV-VIS variety, however, there is a substantial increase in attenuation due to the fiber at wavelengths less than 400 nm (see attached graph from the manufacturer). 

My primary interest is in emissions in the 250-400 nm range.  Is there a way to correct the spectral data to account for the increased attenuation from the fiber at < 400 nm?    

I have recently started a project on Luminescent solar contractor. My design is ready to fabricate but I am facing difficulty in measuring the surface emissions, edge emissions, light trapping efficiency of LSC. Most of the researchers have used an Integrated sphere method for these measurements. Can anyone suggest exact the measurement techniques and procedure. Your kind response is highly appreciated. 

In QE measurement by integrating sphere, the spectral irradiance (W/m2) is transformed in to photon irradiance (cps/m2) by dividing the ordinate data by corresponding photon energy at abscissa. The abscissa is also converted to wavenumber scale prior to such transformation, so that the excitation and emission signals could properly be deconvoluted. In this case, we obtain the photon distribution as a function of photon energy in cm-1. The photon distribution for absorption and emission is obtained by taking the difference with reference scattered signal.

Here my understanding is that the total photon count for absorption or emission is the summation of number of photons at each energy. The value of energy is irrelevant (not sure). I am getting difficulty in obtaining the total number of photons for absorption and emission. In literature, the integration is usually taken to get the area under the curve. Also, the abscissa is mostly wavenumber, but sometimes wavelength. My concern is if we take the integration, it also considers the values at abscissa. I tried to integrate same area curve at different wavenumber range (Origin software), which gives different area values. Please suggest if I am making some mathematical mistakes, and also give opinion on how to obtain total photon count and what should be the unit of abscissa for calculations.

Our institute is in search of a new Phosphoimager.  We currently have a Typhoon that no longer works.  Can anyone give me a recommendation on what works well for them, or is the newest technology out there?   We use it mostly for radioactivity detection, and need it to work over a broad dynamic range.  It seems most systems out there require a 32-bit processor or windows XP, which has given us issues using our old system.  Any input would be greatly appreciated!

I am trying to establish a Gli reporter gene assay in Shh light 2 cells. For that I am using a dual luciferase reporter assay system. However the luminescence values obtained are only in the hundreds. I tried some modifications as well, but nothing helped.

Your suggestions will be of great help to me.

I would like to do circular polarizer-photoluminescence measurements, but I need of a standard sample with a well-known polarization degree. Does anyone know if such a standard sample exists?

Can you suggest some discussions about this?

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