Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
http://www.cyto.purdue.edu/hmarchiv/index.htm
Hello everyone.
ORIGINAL QUESTION:
I am new to the world of flow cytometry and have a client who would like me
to sort bionanoparticles. I have a BD FACSVantage-DiVa DiVa with Turbo
Sort. The bionanoparticles range in size from 50-500nm and are lipid based
so will be stained with Nile red.
I would like to know if I can sort the particles that are <200nm if they are
stained with Nile red? Would it then be possible to sort the ~200-500nm
particles and save the conflicts to get two groups, i.e. ~50-200nm and
~200nm-500nm? The smallest nozzle I have is the 50µ. This nozzle has not
been utilised at our institute before, so could anyone give me an indication
of the frequency and pressure to use the 50µ nozzle at?
There is also a possibility of staining the bionanoparticles with Nile red
and another fluorophore if this would aid the sorting process.
One last question: What filter or bandpass would be best to measure Nile
red emission? I have found conflicting data regarding the emission spectra
of Nile red bound to lipid. Practical Flow Cytometry states that the
fluorescence of the lipid-bound dye is about 550nm and another source
(Invitrogen fluorescence spectra viewer) has the excitation max of
lipid-bound dye at 550nm and the emission at 636nm?
I would appreciate any advice/information.
REPLIES:
(NB I had quite a few replies from people wondering the same thing, mainly what is the absorbance/emission spectra of Nile Red)
* I have one for 60 psi but if speed is not important you may want to go down
to 11. 50u 60psi 89.9khz attenuation off, amplitude 14.3, phase 0, drop delay 19.72
----------------------------------------------
* I would be grateful if you either posted any responses you get or sent them on to me as I am looking at doing something similar soon. BTW I will be using the FACSAria. Nile Red can be excited at 488nm (30% efficiency) and emission is at 630/30 (where you pick up PE-Texas-Red). So the 636 emission max is correct.
You will need to thoroughly clean your lines - I generally find that a solution of Domestos will do the trick plus some additional in-line filters (0.2um). I would also filter everything through 0.2um or even 0.1 um for such small particles.
----------------------------------------------
*You dont have to change the nozzle, as the fluorescence signal will be the differential property you sort on.
Dont know about Nile Red sorry
----------------------------------------------
*Regarding splitting them into two populations based on their size, I have a suspicion it will be difficult to resolve the signal with FSC...might just be lost in the noise of the detector, electronics and the laser. No doubt you'll be popping in the smallest ND filters into the FSC detector to try to see them clearly. Wonder if there are some .5u beads you can buy to see how there signal looks in FSC and if you can distinguish it? Is log on FSC a good idea too? I'd be fascinated to know if you can do it. Keep me posted.
Anymore help (especially about Nile Red ab/em spectra) would be greatly appreciated.
----------------------------------------------
We actually used nile red for lipid / protein differentiation in food emulsions at Colworth. One of the original articles is by Fowler and greenspan in 1985 (www.jlr.org/cgi/reprint/26/7/781) . I actually use it sometimes to stain interfering micelles when measuring nanoparticles below 100nm. However there the fluorescence is mainly orange, but the emission wavelength is very environment dependent. Signal is indeed very weak at that level. To achieve a good signal to noise ratio I play heavily with the concentration and sample flow rate. Like Bis-Oxonol it seems to love the pipe work (that's fairly long in the Elite) and takes a bit to reach a steady state. It is a bit of a hit and miss experience.
As I only got a ~70u orifice I use it and it works fine for things that small. However, the Elite measures in the flow cell, not jet in air..
Good luck
Gerhard
----------------------------------------------
There is an article by Brown et al, Histochemistry v97 p349-354 (1992) that in neutral lipids the emission max of Nile Red is in the 530-560 nm range and in phospholipids the emission max is around 630 nm. This is cited from the original paper by Greenspan & Fowler, J Lipid Res v26, p781-789 (1985), which has curves for excitation and emission spectra of Nile Red in a number of environments. Despite the complexities, NR has been used in a flow assay to measure intracellular accumulation of phospholipids, see: Halstead et al, J Applied Toxicol. v 26, p169-177 (2006). They used the FL-3 channel on a FACScan to measure phospholipids in peripheral blood leukocytes.
With reference to the original question on measurement of NP, the question IMHO is not one of light scatter, but how strongly the particles can be loaded with Nile Red and whether the resulting fluorescence intensity is enough to trigger on using fluorescence triggering.
-Jim Weaver
----------------------------------------------
I do not think that you would be able to sort these particles with standard FACSDiVa.
1/ spatial resolution issue: current cytometers, especially jet-air cell sorters like
DiVa do not give you subcellular 50-500 nm range.
2/ CyFlow-ML high-end cytometer from Partec claims to have a spatial resolution less than
200 nm for scattering and fluorescence of particles, but they have a very sensitive PMT,
not a diode for forward scatter. The sorting speed of this cytometer (if it has a sorting
option) is too low.
3/ with sorting you will have additional issues- noise etc;
From our experience-we were able to sort 1 u beads with standard FACSAria (thank you,
Steve Merlin from BD) w/o PMT on FSC.
You may consider to replace diode to PMT on FSC, but scattering resolution will be still
less than Aria (DiVa is jet-air and less sensitive).
Just my 2 cents,
Natasha
----------------------------------------------
Hello,
I am going from memory here so please be kind.
There is an old paper that describes the excitation and emission characteristics of Nile Red. I believe it is the original paper describing a derivative of lipid stain called Nile Blue (that is not fluorescent.... I think) and the "new" dye is called Nile Red. I have the paper and can provide the reference if you want. Nile Red is virtually non-fluorescent in an aqueous solution and is only fluorescent in a hydrophobic environment. Both the absorption (excitation) and emission is dependent on the polarity of the non-aqueous solvent. In polar solvents, the absorption and emission is red-shifted and in non-polar solvents the absorption and emission is blue-shifted. For cells stained with Nile Red and excited with blue (read 488nm), membranes fluoresce in the deep red (>650nm) due to phospholipids. Endosomes containing neutral lipids such as triglycerides, cholesterol, etc., fluoresce green (~530nm). Cells treated with LDL (low-density lipoprotein) are readily distinguished from untreated cells by an increased green fluorescence since the red fluorescence can be used to normalize for dye loading.
This is an excellent dye and was once used extensively in arterial sclerosis studies on cholesterol deposition using fluorescence microscopy - mostly in rabbits. There were a few publications using Nile Red to study lipid composition of "fat" droplets in milk, but other than that, I have not seen much flow work with it.
Hope this helps but it may be of little help in sorting bionanoparticles. You should see Nile Red fluorescence from the green to the red, provided your nanoparticles are of sufficient mass to bind enough fluorochrome to provide a signal above background.
Regards, TomD
----------------------------------------------
