Reactive Oxygen Species - Science topic

There are many proxies to measure the firm performance of private sector firms, such as ROA, ROE, ROI, ROCE, ROS, etc. How to measure the performance of public sector firms, please? Could anyone suggest something, please? Suggestions, recommendations, and comments are welcome. Thank you

How to calculate fish ROS activity from absorbance? The ROS level was measured by the modified dichlorofluorescein-diacetate (DCF-DA) method. Can anyone guide me with the calculation through excel sheet?

i am coonducting research on wheat to determine salt stress on triticum aestivum while tea residue as mistication agent .want to know how plant respond to stress genetically?

How reactive oxygen species are produced?

Dear researcher

I have obtained my OD at 485 and 522 nm. I don't understand how to interpret the data. Kindly help me to calculate ROS

We want to analyze whether T cells produce ROS under certain pahtophysiologic conditions.

I am looking for information on ROS detection methods that is not a secondary protein change or etc from the ROS presence. Would like to use it in live tissue and it remain stable after PFA and detergents treatment. Thanks in advance.

Facing an issue with H2o2 control experiments with cancer cell line. Could anyone help me.

Thanks

please provide some papers or standardized protocols to check ROS activity in blood samples.

Before neurotoxicity with ethanol, giving an excess of antioxidants (vitamin E, DHA, carotenoids) previous days before intoxication can hinder the ability of the enzymes (because the antioxidants are already dealing with ROS and RNS) to deal with sudden the oxidative stress?

Hello, I need to measure ROS and NO in plasma samples.

I have a fluorescence reader for ROS and reagents for the Griess test for NO.

Do you have a protocol for the preparation of the samples?

I couldn't see much paper where plant breeders use biochemical such as proline content Malondialdehyde (MDA) and dyes such as NBT or DAB( for ROS detection) for screening stress-tolerant accession on a large scale (100-200 which I suppose is possible to do). Are not these methods better than phenotyping grain yield, biomass, plant height, NDVI, LAI, etc?

There is a antioxidant defense system in cell.

Have any signalling pathway or other things can influence ROS level in a extracelluar way?

I was wondering if anyone has documented the generation of reactive oxygen species (ROS) in growth media under light irradiation, with the ROS generated by endogenous photosensitisers in growth media?

I'm curious about what photosensitisers are being utilised here, and what I can do to prevent this in in-vitro photodynamic therapy experiments. Ideally we would like no ROS under light irradiation alone.

For determination of ROS in mitochondrial fraction using DCFDA, How to quantify the concentration of isolated mitochondrial fraction? using nanospectrometer or some other technique ?

If i perform BCA then it will give protein concentration in mitochondria....

But in that protein fraction, i don't think ROS production would b getting accessed.

So, if i hv isolated mitochondrial fraction, then how can i quantify its concentration?

As for DCFDA, i need some specific concentration of mitochondrial fraction (per se: 1mg/ml), so how to determine that concentration?

as in protocol they hv not mentioned anywhere that i need to hv mitochondrial protein fraction (1mg/ml)...they just said: mitochondrial fraction...

so without BCA how can i quantify my isolated mitochondrial fraction easily and quickly so that i can proceed my mitochondrial fraction for further experiments like ROS or membrane potential etc...

kindly confirm

Let's say we have a hypoxic event in the brain which lasts under 0.5 seconds. Is this duration sufficient for ROS formation?

Hello,

I would like to detect ROS using DHE in tumor sample in situ. I have thought of the following scenario:

- to inject 20 mg/kg DHE to mice ip and after 1 hour to sacrifice the mice, take out the tumor, snap freeze, cut with cryostat and visualize under the microscope.

Do you think it is appropriate for detecting ROS within the tissue?

Any comment, suggestion is appreciated!

Thank you!

Eva

Hello everyone,

I am trying to measure reactive oxygen species (ROS) levels of PBS-homogenated brain tissues by DCF assay. I started with control experiments by only using PBS and hydrogen peroxide (H2O2). However, I can not get reliable and consistent values on fluorescence measurement. I tried with 1000, 100, and 10 uM H2O2 with PBS (PBS + 1000 uM H2O2 + DCF) but my values are not consistent between duplicates, for example, 10 uM H2O2 levels are higher than 100 uM H2O2 levels. Besides my controls - they only include PBS and DCF - are higher than 10 uM H2O2. What would be the reason for it? Sometimes I got signals from empty wells as well, does it indicate a problem regarding fluorescence signaling? I use 96-clear well plates and wrap them in aluminum foil to protect them from light as much as possible. I also leave plates at 37 degrees for 30 minutes before reading. Thanks a lot for any suggestions and/or comments in advance.

How to diminish the negative effects of ROS in plant cell?

Any practical idea is most welcome.

I am trying to measure the reactive oxygen species for a Verticillium dahlia mutant using DCFH-DA, can anybody suggests me some protocols how to do this?

thanks in advance

The production of ROS (Reactive Oxygen Species) is toxic to plant cells that deviate or interfere with the normal biochemical pathways. I had targeted quantifying different forms of ROS within specified tissue such as roots and leaves.

I want to use tissue homogenate supernatant (Not tissue) for detecting total ROS by using H2DCF-DA dye. I know it is a cell-based technique and DCFH is a fluorogenic dye that measures reactive oxygen species (ROS) activity within the cell. But would it be possible to obtain the same results from supernatant as all cellular proteins are known to be present in the supernatant?

I am willing to know has anybody tried it before and the procedure they followed.

P.S. Tissue homogenate was centrifuged at 10,000 x g for 15 min. at 4°C. Afterward, the supernatant was stored at -80°C.

Thanks

Hi, I'm searching for a fluorescent mitochondria marker (to be used in live confocal microscopy) that is not dependant of membrane potential nor ROS production. I'll have MitoSox to quantify ROS production but I also want to quatify mitochondria because my treatment could affect ROS production or mitochondria viability. I've read that MitoTracker Green is independant of membrane potential, but the signal can be affect by ROS. I've also found Nonyl Acridine Orange (NAO), which is not affected by ROS, but it doesn't seem clear if it's independant of the potential.

I am using the Abcam ROS assay kit which uses DCFDA and gives reading in fluorescence values measured on a fluorescence plate reader. Does anyone have experience with how to analyze data and plot the values to detect ROS? I am using astrocytes but regardless of that I am concerned with the data analysis part. Thanks a lot in advance, Praveen

Give your suggestions with publication.

I'm trying to mesure total ROS production in bone marrow derived dendritic cells with H2DCF-DA (Invitrogen), but I having some problems.

After differenciated, I plate the cells to 1x10^6/ml and treat them for 18hs with LPS 100ng/ml or left untreated. Next, I stain the cells with the surface marker CD11c-APC and a Live/Dead dye, wash them with PBS and finally incubate with 10uM H2DCF-DA in PBS for 30 minutes at 37°C, protected from light. I wash the cells with cold PBS, resuspend them in PBS-FBS2% and aquire them inmediatelly in a BD FacsCantoII.

The problem is that my untreated cells show a much higher geometric mean that the LPS treated cells.

I've tried different protocols, including resuspending the cells in RPMI-FBS10% before adding the H2DCF-DA in PBS, but I didn't have any luck.

I would really appreciate any help or suggestions.

How can we know the ROS production when we treat bacterial cells with an antibiotic agent?

Hi,

Just a little question about ROS. I measured ROs in untreated and treated (2T) with Ultrasound,

I've observed 2 peaks in DCFH-DA in WT. It's normal? In some experiments, the second peak appears always but variable in intensity. Do normal lymphocytes have a different basal ROS corresponding to different lymphocytes subsets?

the second question in my case, how can I interpreter my data? 2T treatments produce or not rise of ROS? should I normalize my gMFI to cell numbers?

All works report only one peak of ROS, that's for me it's strange

Ps the different count is due to loss of some lymphocyte by treatments.

thanx for suggestions

Dear all!

What is the better way to impose cold /freezing stress to wheat plants. I need see the response of various antioxidants and ROS in wheat after exposing it to freezing/chilling temperature.

When I carried out the active species capture experiments to study the photocatalytic mechanism, I found that the amount of capture agents was different in different work, for example, in a paper, EDTA 1 mmol/L, tert-butyl alcohol (BuOH) 5 mmol/L and p-benzoquinone (BZQ) 1 mmol/L, but in another paper, their amounts are all 1mmol/l. This really confuses me, I don't know how to select the amount of capture agents in my experiments, especially for BuOH.

What do you think of this problem? Is there a general test standard or reference?

Thank you very much for your suggestions and answers.

Hi,

Just a little question about ROS. I measured ROs in untreated and treated (2T) with Ultrasound,

I've observed 2 peaks in DCF-DA in WT. It's normal? In some experiments, the second peak appears always but variable in intensity. Do normal lymphocytes have a different basal ROS corresponding to different lymphocytes subsets?

the second question in my case, how can I interpreter my data? 2T treatments produce or not rise of ROS? should I normalize my gMFI to cell numbers?

All works report only one peak of ROS, that's for me it's strange

Ps the different count is due to loss of some lymphocyte by treatments.

An experienced immunologist maybe could me help.

This dye itself has color of PE-Cy5 and after interaction with the lipid ROS, it emits green fluorescence. I have also found that this dye also interferes with the PE, PE-Cy7 channels somewhat. So I was wondering if there is an alternative to this dye? (Also, why is this the standard dye for ferroptosis related lipid peroxidation?)

Can activating neutrophils with IL-8 at different concentrations show different responses?

I want to detect ROS production in cells but spectrophotometrically, using an UV-visible spectrophotometer. As where I work we don't have a fluorescence reader (as used with the h2DCFDA probe), I wonder if you could recommend which non-fluorescent dyes or reagents can be used for this purpose.

Thanks in advance!

I'm using abcams kit DCFDA/H2DCFDA CELLULAR ROS ASSAY KIT to measure ROS in human peripheral blood mononuclear cells by flow cytometry, but I'm having some trouble with positive control (TBHP), I have used different concentrations (50, 100 y 250uM) and negative control.

I would like to use amplex red to quantify ROS from coral tissue samples that have been snap-frozen. Is amplex red the best way to measure ROS. I will be measuring ROS from tissue samples rather than from Symbiodinium cultures. TIA

We are standardized the ROS detection in PBMC culture using DCFDA assay. For establish cells controls conditions, we are cultured humans PBMC during 24 hours, and 4 hours before to end the cells culture, we treat some cells with 100µM of TBHP in 10% FBS during 4 hours. After that, we takes cells with or without TBHP, treat with 2.5µM of DCFDA during 30 min, and analyzed in Flow Cytometry. Ours results are confused, because the DCFDA signal between positive and negative control are similar. Thinking, in the problem would be the concentration of TBHP; we increased the concentration to 250µM and the DCFDA signal in the positive control were less than negative control.

I would be grateful for any suggestion.

Well, we read a lot about how in vitro manipulation of oocytes can induce stress and therefore reduce their potential or as people call it "quality". I've been looking into the studies mostly, they focus on the oxidative stress and reactive oxygen species relation with calcium regulation. Other studies investigated the heating as a stress factor. Well, how they established that this oocyte is healthy and this one is stressed? On what basis? What kind of methods they used? Are the polscope data useful?

I want to measure ROS synthesis in cells grown in transwell. I cannot measure fluorescence directly in the transwell because of the high so I have to remove the cells. One option is to use trypsin but I guess if someone has used another method. I use DCFDA kit (Abcam). Thank you

I treated SK-N-SH cells with 1mM H2O2 and 1mM H2O2+antioxidants (3 different mixtures) and measured the mitochondrial membrane potential (ΔΨm) with 500nM TMRE. As far as I know, H2O2 and ROS generation depolarizes the mitochondrial membrane so we expect lower values in TMRE signal rather than the H2O2+antioxidants mixure. I repeated the process 3 times and the result is the same.

Im trying to get Velocity in order to control underwater swarm ROV. I can use the acceleration and to Euler integration but the error will be accumulated over time. As we are having a swarm (many small robots) Im limited with sensors on board. So in order to get more accurate Velocity estimation was thinking of sensor Fusing of IMU and odometry. As don't have any odometry sensors came to some out of the box solution.

So as I have access to the motor (thruster- propeller) signals and the robot can move to maximum and constant speed. Means the rotation of the propeller have a maximum limit and when they reach that limit they can maintain that maximum. So was thinking of some mathematical model that can be used to provide me with odometry and than use some of the ROS packages for sensor fission(robot_lokalization or hector_pose_estimation) to get accurate Velocity

So I need a help in that mathematical model how to get odometry from the motors-thrusters. Any help with that and possible ROS node (C++) implementation?

Thanks

Hi, could you teach me if anyone has experience in detection of Reactive oxygen species?

I would like to detect ROS in the cytosol and in the mitochondria separately.

Mitosox can detect mitochondrial ROS specifically.

I wonder that ROS detected by DHE is contained mitochondrial ROS?

I am working on lung inflammatory disorder which is marked by oxidative stress. In order to measure mitochondrial ROS in bronchoalveolar lavage fluid (BALF) sample procured from mice, I am planning to utilize mitoSOX probe. I have a query if we can directly use mitoSOX on BALF cells and measure the fluorescent intensity through flow cytometry? OR

Do we need to use antibodies (along with mitoSOX) against specific markers of inflammatory cells involved in lung inflammatory disease?

To my understanding there is a kind of "black box" concerning the development of the disease itself: from its onset to the point when patients get hospitalized. Furthermore some potentially useful information coming from MDs is rarely taken into account. They often describe their patients as suffering from high altitude sickness. High altitude sickness is induced by a lack of oxygen.

During the first days of the disease the lungs are not yet damaged. The destruction of beta-hemoglobine has already been discussed, but this is seldom mentioned when we talk about different ethnic groups; modifications of hemoglobine protecting against Malaria might be the reason for their higher mortality.

A lack of oxygen is called hypoxia. Hypoxia induces the formation of ROS and RNOS. They can directly damage cells, amplify the inflammatory reaction and they act as second messengers in the immune system. It is even known they are related to human coronaviridae, but their role in COVID-19 is poorly taken into account.

Hi, I'm trying to study the impact of reactive oxygen species on whole blood with respect to how it may induce proteolysis on an intracellular protein in PBMC's in a simulated extracorporeal circulatory model. Instead of exposure of PMBC's to hydrogen peroxide, I need to mimic the actual inflammatory environment such as cardiopulmonary bypass and therefore need whole blood. Trialed H2O2 diluted down to 0.05% without much success. Any suggestions would be much appreciated.

Hello everyone

I do not completely understand the theory of processing the data of flowcytometry DCFH-DA based ROS kit.

What do "Geo Mean" and "M2" refer to? Which one should I use as a ROS indicator?

Noting that they had given me conflicting results.

Any advice will be appreciated.

Hi

I have IMU sensor that gives me the raw data such as orientation, Angular and Linear acceleration. Im using ROS and doing some Gazebo UUV simulation. Furthermore, I want to get linear velocity from the raw IMU data. If I do integration over time there will be accumulated error and will not be accurate with the time when for example the robot makes turns.

So If I use

acceleration_x = (msg->linear_acceleration.x + 9.81 * sin(pitch)) * cos(pitch); acceleration_y = (msg->linear_acceleration.y - 9.81 * sin(roll)) * cos(roll);

So integrating linear acceleration is very bad,

Velocity_x= Velocity_old_x+acceleration_x*dt;

because integrates the acceleration without taking into account any possible rotation of the sensor, which means that the results will probably be terrible if the sensor rotates at all.

So I need some ROS package that takes into account all this transformation and gives me the most accurate estimation of the linear velocity. Any Help? Thanks

I would like to use 2′,7′-Dichlorofluorescin diacetate to assess the ROS production in a549 cells growing in a 96-well microplate.

I have found protocols similar to the following in various papers, but I still have questions about the procedure.

Any suggestions or advice would be welcome.

Reactive oxygen species was responsible for detection of Ascorbic acid, dopamine, uric acid and glutathione by colorimetric method?

In those case majority of nanozymes was OH radical and some super oxide anions. how selectively detection in the presence of ROS. the above mentioned biomelcules have selective shown in numerous papers but same reactive oxygen species? How that same ROS behave different for biomoelcule?

please suggest some valuable answer.

I would like to measure mitochondrial ROS in different human fibroblast cultures, and I have in total 30 different cultures. Since superoxides are too easily converted, one tactic is to incubate one cell line, then measure. But with 30 cell lines, this would take quite a long time, and is the results then reliable. So the question is, can I set up let's say 5 cell lines, incubate with MitoSox, and then fixate the cells som how keep the fixed cell in the dark and then analyze? Does anyone have experience with this?

cheers,

Recently, I am performing the intracellular ROS detection by DCF-DA method in SHSY5Y cells through flow cytometer. But, I found a high fluorescence intensity for the untreated control stain with the probe DCF-DA at 10 micro molar for 30 min, 37 degree celcius in dark than that of the ros inducer i.e. rotenone. Why is it happening like this? Please tell me.

I have done simulation of single drone using hector quadrotor for indoor and outdoor environments using ROS kinetic and Gazebo7. Please help me to do the simulation for drone swarms using same package.

Hi everyone. As GO or HA/FA itself produce ROS radicals if we treat them lonely with UV light but GO and HA/FA in NaCl suspensions do not produce ROS under UV light. What may be the possible reasons for it? Is quenching power of GO due to pie-pie stacking or competition of GO and HA/FA for ight absorption may be the leading mechanism for it? Or reduction of GO under UV promote the aggregation of GO due to which light absorption decrease and ultimately no ROS generation? Or there may be any role of NaCl?

Looking forward for a fruitful discussion.

Regards

We are looking to do a time dependent mtROS measurement after MPP⁺ treatment given to cells. Can anyone suggest if pretreatment with MitoSOX ( followed by washing) could be given before cells are treated with MPP⁺. I have found papers following this procedure for the total ROS estimation using DCFDA but couldn't find using MitoSOX.

Hello everyone,

I am asking very general question, but i would appreciate if someone helps me. I want to use ROS for manufacturing process (Not robots) as a middleware that can create simulated Cyber physical system environment to enabling the interoperability of different nodes in real time. Before start involving in ROS, i would like to ask you below questions.

Thanks a ton

I want to determine ROS levels in freshly isolated and/or snap-frozen primary leukemic cells. Any idea? Please suggest references, if possible.

Is there any photosensitizer that can generate different types of ROS upon different light irradiation? I understand my question is kinda confusing so I added an example below this.

(E.g., When the photosensitizer is irradiated by white LED light, it generates singlet oxygen mostly. On the other hand, when it is irradiated by green LED light, it generates radicals mostly.)

I want to find something about detection of ROS content produced by different light irradiation to the photosensitizer.

If you guys can find any journals that can answer my question, pls let me know! thank you.

ROS (reactive oxygen species, DCFH-DA, DHE), TAC (Total antioxidant capacity). ROS measured using flowcytometry but TAC measured by a kit and elisa reader.

I am treating my cells with a drug that generates about 20-40 uM H2O2. I see a profound activation of gamma-H2AX but no/very low levels of corresponding DNA damage in comet assay.

I am trying to find out if the induction of gamma-H2AX is somehow related to the generation of H2O2 in my drug treated cells.

Hello

It is been a long time I have been trying to stimulate vascular smooth muscle cells (MOVAS) for ROS generation. I am using DCFDA assay for ROS detection....

Everything I tried, has failed to stimulate it! LPS (short and long incubation time), high and low concentration!, Iron (Fe3+), glucose (25 mM 50 mM etc), Beta-glycerophosphate (up to 10 mM).... none works!

Does anybody have any idea what is happening?

Extremely frustrating cycle of trial and failure !!!

I used MitoSox red dye for the measurement of ROS levels in pancreatic cancer cell lines.

I'm working on cell apoptosis now, and I really need a stable inducer of intrinsic apoptotic/ROS-related/mitochondria-related apoptosis. I've tried H2O2 and ABT(chemical drugs), but they both have unstable results. So if anyone has any suggestions, it would be very nice.

Thanks :)

Does anyone have an idea related to commercial availability of any NIR probe for in vivo detection of ROS?

In Egypt, ambient temperature can remain consistently high for extended periods of time and sudden recurrent hot and humid waves that have more harmful effects. So poultry production suffers significant losses every year because of heat stress, leading to economic losses to the poultry farmers. During these periods temperatures approached 40˚C most of the time and humidity reaches 75%. A temperature above 30˚C represents a heat-stressed condition for birds and is one of the most common stressors that affect poultry production criteria where the ideal temperature for broilers is 10-22˚C to get optimum body weight and 15- 27˚C for feed efficiency.

Heat stress is principally important in intensive poultry operations especially in broilers lines because their higher production performance and feed conversion efficiency make today's chickens more susceptible to heat stress than ever before.

High mortality, decreased feed intake, lower body weight gain and poor feed efficiency are common adverse effects of heat stress often seen in meat-type poultry flocks. In addition, heat stress increases lipid oxidation as a consequence of increased free radical generation, a condition that enhances the formation of reactive oxygen species (ROS) and induces oxidative stress in cells. Antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) play a vital role in protecting cells from the harmful effects of ROS. Synthesizing these enzymes is an important regulation, in terms of animal response to stress conditions. However, this response will be effective only if cofactors such as Se for GPx and copper, zinc, and manganese for SOD are available.

I am trying to detect ROS in some breast cancer cell lines, including MDA-MB-231, MCF-7 and 4T1. I found out that ROS in some cell lines decreased after the incubation with H2O2, while in some cell lines ROS increased. I used DCFH-DA as prober. I just feel confused about the results. Could someone provide some clues?

I used DCFH method to measure reactive oxygen species in the atmosphere, using hydrogen peroxide as the standard solution, and found that when hydrogen peroxide is not added, the fluorescence intensity is very large, I do not know what is the reason?

I am interested in determining the intracellular ROS concentration but we do not have a fluorescence spectrophotometer. Could someone recommend an alternative technique where the determination is carried out in UV-visible and is adaptable to a 96-well plate.

Thank you

Hi!

I am looking for a fluorescente probe to evaluate ROS levels in specific subregions of mouse brain. I tested DHE but the staining was totally unspecific!!!

To test DHE before the experimental conditons, I tested the compound in acute brain slices treated with Rotenone: 2um, 1um, 500nm, 200nm (3min) followed by 15 min wash.

The control slice was not treated with Rotenone.

I fixed the slices in PFA 4% over night, sunked in saccarose 30% and the cutted in 30um slices.

DHE staining protocol:

-staining: 5um, 15', RT

- wash: PB, 2x1min

- wash: H20, 1min

Unfortunatelly, all neurons of all conditions (also the control one!) showed high fluorescent signal!!!!

Thus, I stained 30um slices from a perfused brain (from naive mouse) and I checked an intense and unspecific fluorescent staining in all neuronal population also in this condition!

How can I solve this problem?

Can someone suggest me a different probe to analyze ROS in mouse brain?

Thank you for your help!!!

I would like to label some fixed cells/platelets with CM-H2DCFDA (General Oxidative Stress Indicator) dye . I would like to know if this is possible in fixed cells. Does the dye bind covalently to intracellular targets? Do fixed cells even have ROS?

According to ThermoFisher Scientific:

CM-H2DCFDA is a chloromethyl derivative of H2DCFDA, useful as an indicator for reactive oxygen species (ROS) in cells. This indicator exhibits much better retention in live cells than H2DCFDA. CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus facilitating long-term studies.

Thank you.

I incubated E.coli (10^8 CFU/ml) with DCFH-DA (final concentration 1 mM) in dark for 30 mins. Then I scanned the sample for an excitation wavelength of 485-495 nm and an emission wavelength of 500-600 nm. But I did not find any significant emission peak (as shown in the attached figure).

Please advise me on how I can improve the ROS detection by DCFH-DA.

My research is looking to detect cell activation (specifically PAMs) through ROS measurement.

Hallo everyone,

I am trying to decrease ROS by adding glutathione (reduced) in human retinal endothelial cells under hyperoxic conditions. Unfortunately a lot of cellular ros assay kits like H2DCFDA are based on fluorogenic dyes which react with glutathione. Does anyone have experience or an idea how to measure oxidative stress and/or ROS based on reactions without interference with strong ROS scavenger like glutathione?

Thank you in advance

Justus

I`m trying to measure increased ROS levels, caused by ATP in RAW macrophages, but I see no difference versus control. I used HBSS, 300 μM - 3 mM range of ATP and 3 hours - time of incubation. After that I included LPS priming (100 ng/ml). It makes result only with 100-300 μM, but I can`t get good repeatability and cell viability (cells stop adhering and are washed out of the plate). Сan anyone advise a suitable protocol?

Dear all,

These days I had a problem in choosing the mode of microplate mode. I need your help to know if what I did is right.

My goal: To use MitoSOX (Ex/Em: 510/580 nm) to measure mitochondrial ROS in macrophages.

You can find Structure & Spectrum here https://www.bioblast.at/index.php/MitoSOX

According to Reference paper ,

"The fluorescence intensity of MitoSOX was measured by a microplate reader...at excitation and emission wavelengths of 510 and 580 nm, respectively."

So, I switch on my lovely Tecan plate reader, open "Magellan" software, chose "Fluorescence Intensity" ----"Wavelengths"...then...I chose "Fixed Wavelength" mode and set the "exciation wav" and "Emission wav" into 510 and 580. but...Is this right?

By the way, in which condition do you recommend "excitation scan" mode or "emission scan" mode rather than "Fixed Wavelength" mode ? As a beginner, I really do not know the difference now.

I summarised my setting in the attached jpeg and pptx.

Thanks.

I have exposed microalgae cells to temperature stress and measure the ROS level using DCFH, but I found that the DCFH was significantly lower in stress-treated samples compared to control. How is this possible?