[Cytometry] microvesicle analysis

Ed Podniesinski flow8775 at roadrunner.com
Sun Feb 2 14:03:46 EST 2014


Uttara,

We conduct microvesicle experiments on a Fortessa cytometer analyzer but not
currently on our sorters.
We use a separate sheath tank (keep it clean)  coupled to a .1u sheath
filter. We use Leinco sheath fluid.
I also clean the system for 20 minutes with BD FACS Clean, already
prefiltered, no fluid filter. This is a core facility user cytometer by many
users.
After the FACS Clean the instrument is washed out with reagent grade water
through its own .1u fluid filter, 30- 45 minutes.
I remove the fluidic side panel to jack into the fluidic as close to the
sheath fluid input as possible with minimal tubing lengths. 
There is a connector I disconnect which eliminates tubing going to our HTS
and other possible sub-micron particle noise generating runs.

To get our initial settings we run Spherotech NFPPS-52-4K Nano Particle kit,
FITC fluor.
It is best to start with a FITC Trigger on these beads before you try a
Scatter trigger.
First each particle size is run individually, 1.38u, next .88u, .45u lastly
,22u.
The scatter is optimized to obtain plots of each bead population.
The final test is to combine all the bead sizes into one tube and run.
Filtered sheath from the .1u filter is used to prep the nano particle
samples.
Flow Rate on low with Vernier flow adj to max then backed off to where
events are still stable, usually to min adjustment. 
A FITC vs Time plot helps in flow rate stability check.
Region boxes on the scatter plots approximate bead size to biology, but is
approximate.

The settings we came up with for 50 mw 488nm laser excitation:

FSC  638 LOG
SSC  365 LOG
FITC 467 LOG
Bi-Exponential setting on plots

Scatter trigger we use once FITC trigger obtains nice populations:

Switch from FITC Trig to:

Dual Trigger using OR
SSC  @500 OR
FSC @ 5000 OR

The intent is to use Fluorescence trigger but the scatter trigger is used
while running .1u sheath as this sample gives us an indication as how clean
the system is.
There are times we toggle over to scatter trigger with confidence after
reviewing the system quietness while running .1u filtered sheath. 

The FSC is a photo diode which results in elongated FSC detected events at
these sizes, limited dynamic range compared to a PMT. We don't have the FSC
PMT option.
The SSC detector uses a PMT which results in excellent resolution down to
.22u. The populations are nicely resolved.

Hope this helps,

Ed Podniesinski

http://rpciflow.org




-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Uttara Chakraborty
Sent: Tuesday, January 28, 2014 6:33 AM
To: Cytometry at lists.purdue.edu
Subject: [Cytometry] microvesicle analysis

Dear Flow Community,
I have some queries regarding microvesicle analyses by flow.

Is it possible to observe distinct scatter properties of microvesicles in
FACS Aria III. If so, what are the parameters that should be taken care of
and what size range will they fall into.
Do they have any RNA content when they are shed off.
Is it possible to use RNA specific dyes to exclude exosomes from my
population of interest in the same size range.
Eagerly awaiting your suggestions and advice.
Thanks in advance.
 Uttara
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