antibody calculation problems

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raves
raves's picture
antibody calculation problems

I am having some difficulty with this calculation of antibodies for facs analysis. The volume of cells i start with is dependent on the number that are present, e.g 1,000,000 cell is approx 40ul and 10,000,000 cells in approx 100-120ul volume. if my stock is 2mg/ml and i add 1ul of this stock to my 1,000,000 (40ul) cells is the final concentraion of antibody in my cells 0.2ug?

If it is 0.2ug and i want to have the same concentration of antibody in 10,000,000 cells how much (ul) of my antibody stock do i add to my cells (which will be a volume of 100-120ul)

marcus muench
marcus muench's picture
concentration is 2 ug/ml

concentration is 2 ug/ml

you need 3ul for the 120ul volume (3 times the volume, thus 3 times the Ab).

Dave@AbDSerotec
Dave@AbDSerotec's picture
Actually, the concentration

Actually, the concentration would be ~50ug/ml (48.8ug/ml).

Here's the calculation:

2mg/ml = 2ug/ul
2ug/ul x 1ul = 2ug

So you added 2ug of antibody into your 40ul of cells (your total volume is now 41ul).

2ug/41ul = 0.0488ug/ul = 48.8ug/ml

I hope this helps!

samm
samm's picture
You need to remember the

You need to remember the (reqd/given conc)*final vol formula (c1v1=c2v2) - that makes these calculations much easier.
That said, Dave has it right - it would be ~50 ug/ml.
It is very important to use the same amt of Ab in a fixed vol for x number of cells whenever you do a FACS assay. For most population expression analyses, 250k-1 million cells are enough, however if you are seeking low abundance markers like a specific TCR in the population, you'll of course need much higher cell numbers, and will need to scale up both Ab amount and volume appropriately. You can easily stain 10 million cells in 100 ul volume, with appropriate scale-up in Ab amt.

marcus muench
marcus muench's picture
I clearly should stop

I clearly should stop answering dilution problem questions late at night. Dave is absolutely correct.

marcia (not verified)
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Carson you're great! Don't

Carson you're great! Don't neglect your sleep. Brilliant minds need sleep too.

samm
samm's picture
Don't forget the caffeine

Don't forget the caffeine either! ;)

My neurons seem to be doing their thing in fits and starts, so you can commiserate with me...

raves
raves's picture
Thankyou to everyone for your

Thankyou to everyone for your help with these calculations. Its very much appreciated and makes perfect sense

Thankyou