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Counting beads binding to RBCs


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#1 biology_06er

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Posted 24 February 2016 - 03:13 PM

Hi guys,,

 

Sooo...I have been using counting beads to quantify the concentration of RBCs in a mouse. However, when I count 1000 beads/sample for example, half of my bead population binds to my RBCs. Initially, I was gating out beads from my control "just bead" sample on Forward/Side scatter. Therefore, in my calculation I was using 500beads instead of 1000. When I include 1000 beads in my calculation, my concentration of RBCs, halves which makes sense because my bead volume is increased but RBC number is staying the same. However, my supervisor thinks that if I use 1000 beads in my calculation my conc. should double. He explained it, it made sense, but now I go back to my workings it doesn't...anyone able to explain a) have your counting beads ever bound to RBCs and B) re-explain what my supervisor means....hopefully I make sense!

 

Cheers,

biology_06er



#2 biology_06er

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Posted 24 February 2016 - 04:50 PM

Another question, to check if my maths is correct!! The calculation people use for absolute count is: (#cells counted in population targeted/beads counted)*bead concentration

 

I have been using the calculation originally used by my supervisor. Example: I make up a master mix of buffer/beads and resuspend my sample in 500uL of this buffer. The final concentration of the beads in this master mix is now 10.21beads/uL (stock vial is 10.21/uL and I take 160uL of this, add to 16mL buffer). I have collected 5uL of blood and into 500uL buffer so a 100x dilution.

 

Then I collect say 1million events in targeted population and my beads in my gated sample are 500. So the volume of beads I have sucked up is 500/10.21=48.97uL. Therefore, 1mill events/48.98*100 DF* 1000 = cells per mL

 

Hmm, so I think I might have understood my first question...if my bead concentration from the vial is 1000beads/uL. And in my population I am saying I am collecting 500beads in my gate but my total bead number is 1000 (including the ones bound to RBCs) and the number of RBCs I have collected is 1,000,000. Therefore the volume of beads I am collecting is 500/1000=






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