Cytokine concentrations helpppppp

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techbio
techbio's picture
Cytokine concentrations helpppppp

 Hi,
I will use cytokines for the first time. I have 10µg of a cytokine and I will suspend it in 1ml of PBS, so I will have a concentration of 10µg/ml.
I need different concentrations of this cytokine: 10ng/ml, 100ng/ml, 1ng/ml  and 0.1ng/ml…
I thought that I should start by preparing a solution of 10µg/ml then from this solution I will take 1µl that I suspend in 999µl of PBS to get a concentration of 10ng/ml.
For a concentration of 100ng/ml I will take 10µl from the solution of 10µg/ml that I add to 990µl of PBS.
Please is that correct? If it’s not how should I proceed?
Thank you very much 

vanshita
vanshita's picture
hello,

hello,
your  original stock  conc. is 10ug/ml or 10,000ng/ml. and you need to dilute it 100 times for  100ng/ml,1000 times for 10ng/ml, 10,000 times for 1ng/ml and 100000 times for 0.1ng /ml for whc you need to take very less volume from original stock where error of chences increase due to less volume.
   
  so the best way to make all these concentration  dilute your original stock 100 times( take 10ul from stock 10ug/ml and  suspend in 990 ul of PBS)  nw final concentration of your stock is 100ng/ml or .01ug/ml. Now you can do serial dilution use 100ng/ml dilute it 10 times to get 10ng/ml,100 times to get 1ng/ml and for 0.1ng/ml dilute 1000 times.

techbio
techbio's picture
 thank you very very much, i

 thank you very very much, i will take your advice
thank you so much

marcus muench
marcus muench's picture
 I agree that you should make

 I agree that you should make the most accurate measurements possible with the instruments you have at hand.  Note a 2ul pipetteman is better than a 20ul etc.  It is better to do serial dilutions with accurate volumes than one giant dilution in one step.

Also, remember that as you dilute you protein, it is best if you use some buffer or medium with some other protein in it such as BSA.  At low concentrations of protein, you may have the protein sticking to your tubes or loosing activity because a simple saline solution is no where near physiological.

techbio
techbio's picture
I decided to do tow main

I decided to do tow main dilution from the stock solution 10µg/ml
The first I dilute 100 times that means that I will take 10µl from the stock solution and I will add 990µl of PBS and I will get a final concentration of 100ng/ml, then I will dilute this solution 10 times to get a concentration of 10ng/ml, for that I will take 100µl from the solution of (100ng/ml) and I will add 900µl of PBS
The second I will dilute 10 times the stock solution, so I will take 100µl from the stock solution and I will add 900µl of PBS, I will get a final concentration of 1000ng/ml
 
So if I do a cell culture using 10ng/ml of the cytokine:
CiVi=CfVf
100ng/ml *X=10ng/ml*200µl (volume that I will put in the well)
So X=2µl  so I think that I have to take 2µL from the solution of 100ng/ml
 
Is that correct?
Yes  I will use serum 

marcus muench
marcus muench's picture
 "So if I do a cell culture

 "So if I do a cell culture using 10ng/ml of thecytokine:
CiVi=CfVf
100ng/ml *X=10ng/ml*200µl (volume that I will put in the well)
So X=2µl  so I think that I have to take 2µL from the solution of 100ng/ml"

I'm not sure I followed all that, but if you want 200ul of 10ng/ml final and your using a 100ng/ml stock them you need 20ul cytokine.

techbio
techbio's picture
you are right i put 100ng/ml

you are right i put 100ng/ml instead 1000ng/ml 
so 
1000ng/ml *X=10ng/ml *200µl

regarding the dillution is that correct? even if it sounds simple  but i prefere to be sur 
thank you very much for your responses, i really appreciate it. 

marcus muench
marcus muench's picture
 yes that is a 1:100

 yes that is a 1:100 dilutions, so 2ul in 200ul final.

techbio
techbio's picture
 thank you very muchfor your

 thank you very muchfor your help, now i am rassured 

Rajeshwari patel
Rajeshwari patel's picture
Yes I support vani,

Yes I support vani,

one more point I want to add is that You need to add BSA in your PBS because cytokines  are  unstable
and all aliqutes sould be very small so you can use for single use.
Best of luck
 
Rajeshwari

scien007
scien007's picture
 I am doing this also for the

 I am doing this also for the first time. I need 1, 10 and 100 ng concentrations of LPS. 
My stock solution is 1 ug/ml. The final volume is 200 ul per well. 
Can I add 20 ul + 180 ul of PBS and serial dilute this?
Thanks

marcus muench
marcus muench's picture
Yes your 1/10 dilution will

Yes your 1/10 dilution will yield 100ng/ml, but remember if you remove 20ul for the next dilution that you will end up with 180ul final volume.  if you dilute it back to 200ul then your concentration will be incorrect.  

scien007
scien007's picture
 Would you suggest this? 

 Would you suggest this? 

Stock 1 ug/ml = 1000 ng/ml

C1V1=C2V2

Making the 100 ng solution from stock
1000 ng x C1 = 100 ng x 200 ul
V1 = 20 ul, so add 180 ul PBS

Making the 10 ng solution 
100 ng x C2 = 10 ng x 200 ul
V1 = 20 ul, so add 180 ul PBS

Making the 1 ng solution 
10 ng x V1 = 1 ng x 200 ul 
V1= 20 ul in 180 PBS

Thanks 

marcus muench
marcus muench's picture
 Your Explantation of The

 Your Explantation of The math is a Bit Off.    Nonetheless, 20ul added to 180ul is a 1/10 dilution and if you do this three Times in a Row then you have it.