@Ahemed Enam - Thank you for writing to us; we apologise for the mistake. And, it is a typo error, while voicing the presentation we didn't notice it. Hence, the video visitors will make out the dilution part on themselves; didn't elaborate on it too. Please follow the Trolox dilution as follows: 2 mM Trolox Stock Solution: Dissolve 0.050 g in 100 ml of working Phosphate buffer. From Trolox (2 mM)stock prepare(6.25 12.5; 25; 50 and 100 μmol) of standards each of 1 ml. 6.25 μmol = 3.12 μL in 1 mL PBS 12.5 μmol = 6.24 μL in 1 mL PBS 25 μmol = 12.50 μL in 1 mL PBS 50 μmol = 25.00 μL in 1 mL PBS 100 μmol = 50.00 μL in 1 mL PBS Note here there is no other diluent than PBS.
Your are welcome .. plz subscribe to our channel and also spread a word among your professional mates to watch, subscribe and to give feedbacks to our videos..
if we are using replicates of each dilution of samples then how to calculate Net AUC? Can we take average of AUC of each dilution? Thank you so much for your video
You have considered different dilutions od same sample and therefore you took average but whaht if i have 16 different sample types of same dilution? Can you please explain the calculation for that case.
I believe you are asking questions on the "calculation part of the presentation". The tested sample solution volume is 375uL, taken from a test sample of 1mL. And we know 1mL = 1000 uL. Now, if we consider the 375uL of used test sample is present at 2.66 fold in 1mL of the test sample. (multiply 2.66 with 375 = 1000uL = 1mL).
We can calculate the ORAC concentration of samples in both ways, Firstly as the average of different fold samples as mentioned in the slides and Secondly as individually multiplying net Auc (but not the average net auc) to its respective sample by type 1 and type 2 calculations.. But, after doing the separate calculations for the different dilutions, then you should sum and average again the ORAC VALUE and will be same as the average as in the slides.. Note: your ask is that after averaging the samples and getting average, we suppose to multiplying it to dilution factor and it is wrong.. When you average you are making a common dilution and common net auc to whole dilutions so the calculation ll give common ORAC value results.. Hope i have answered to your both questions.
thank you for writing to us. I believe you are asking about phosphate buffer. Yes Once, the solution is prepared at the required pH. Then can be used for long as the pH remains the same. Self-made salines can be used for up to a month or till it remains good. shelf life for PBS products is about 1 year at 20^C no chemical reaction would undergo in PBS and pH should remain stable, as a least energetic state has already been reached. If you see any changes in transparency or your PBS solutions become turbid, they should be discarded.
@@LEARNERSBIGBOOK I have another question, I have fluorescein from Zigma, do I need to add a salt like ammonium persulfate? or can I go ahead to prepare my stock just with the fluorescein because in this case, I don't have sodium fluorescein.
Fluorescein is an organic dye, not soluble in water or many organic solvents, but its disodium salt (fluorescein sodium, uranine, D & C Yellow ) is highly water-soluble. Molecular Formula: C20H12O5 * - Preparation Instructions; This product is soluble in 1 M NaOH (50 mg/ml), with heating. It is insoluble in water, benzene, chloroform, and ether. * It is also soluble in hot alcohol and glacial acetic acid solutions. Note: try dissolving the direct fluorescein C20H12O5 in buffer PBS, as the buffer has potassium has to get reacted to get bonded. If not you can use the above preparation methods.
How can you get to prepare 2 mM trolox soln that dissolve 0.074 gram in 100 ml?
@Ahemed Enam -
Thank you for writing to us; we apologise for the mistake. And, it is a typo error, while voicing the presentation we didn't notice it.
Hence, the video visitors will make out the dilution part on themselves; didn't elaborate on it too.
Please follow the Trolox dilution as follows:
2 mM Trolox Stock Solution: Dissolve 0.050 g in 100 ml of working Phosphate buffer.
From Trolox (2 mM)stock prepare(6.25 12.5; 25; 50 and 100 μmol) of standards each of 1 ml.
6.25 μmol = 3.12 μL in 1 mL PBS
12.5 μmol = 6.24 μL in 1 mL PBS
25 μmol = 12.50 μL in 1 mL PBS
50 μmol = 25.00 μL in 1 mL PBS
100 μmol = 50.00 μL in 1 mL PBS
Note here there is no other diluent than PBS.
So easy to follow up with such complicated topics sir!
Thank you
Thank you Biozee ...
@@LEARNERSBIGBOOK can you provide pdf format of this procedure?
Its really a good presentation with complete info...
Thank you so much for the complete information.
Your are welcome .. plz subscribe to our channel and also spread a word among your professional mates to watch, subscribe and to give feedbacks to our videos..
if we are using replicates of each dilution of samples then how to calculate Net AUC? Can we take average of AUC of each dilution? Thank you so much for your video
You have considered different dilutions od same sample and therefore you took average but whaht if i have 16 different sample types of same dilution? Can you please explain the calculation for that case.
How did you get the 2.66 fold of 375uL
Thank you for writing to us.
can you elaborate on your question? please.
How did you get 2.6 fold please tell us.
I believe you are asking questions on the "calculation part of the presentation".
The tested sample solution volume is 375uL, taken from a test sample of 1mL.
And we know 1mL = 1000 uL.
Now, if we consider the 375uL of used test sample is present at 2.66 fold in 1mL of the test sample. (multiply 2.66 with 375 = 1000uL = 1mL).
@@LEARNERSBIGBOOK Thank so much I understood
You are welcome .. if u have any more doubts plz feel free to ask..
How can you average different dilutions as one ?
and what happened to the dilution factor? shouldn't you multiply the final value with its respective dilution factors ?
We can calculate the ORAC concentration of samples in both ways,
Firstly as the average of different fold samples as mentioned in the slides and
Secondly as individually multiplying net Auc (but not the average net auc) to its respective sample by type 1 and type 2 calculations..
But, after doing the separate calculations for the different dilutions, then you should sum and average again the ORAC VALUE and will be same as the average as in the slides..
Note: your ask is that after averaging the samples and getting average, we suppose to multiplying it to dilution factor and it is wrong..
When you average you are making a common dilution and common net auc to whole dilutions so the calculation ll give common ORAC value results..
Hope i have answered to your both questions.
Can I use already made PBS of pH 7.4 for this assay?
thank you for writing to us. I believe you are asking about phosphate buffer. Yes Once, the solution is prepared at the required pH. Then can be used for long as the pH remains the same. Self-made salines can be used for up to a month or till it remains good.
shelf life for PBS products is about 1 year at 20^C no chemical reaction would undergo in PBS and pH should remain stable, as a least energetic state has already been reached. If you see any changes in transparency or your PBS solutions become turbid, they should be discarded.
Thank you very much for your response
You are welcome..
@@LEARNERSBIGBOOK
I have another question, I have fluorescein from Zigma, do I need to add a salt like ammonium persulfate? or can I go ahead to prepare my stock just with the fluorescein because in this case, I don't have sodium fluorescein.
Fluorescein is an organic dye, not soluble in water or many organic solvents, but its disodium salt (fluorescein sodium, uranine, D & C Yellow ) is highly water-soluble.
Molecular Formula: C20H12O5
* - Preparation Instructions;
This product is soluble in 1 M NaOH (50 mg/ml), with heating. It is insoluble in water, benzene, chloroform, and ether.
* It is also soluble in hot alcohol and glacial acetic acid solutions.
Note: try dissolving the direct fluorescein C20H12O5 in buffer PBS, as the buffer has potassium has to get reacted to get bonded. If not you can use the above preparation methods.