LacZ detection by FACS with FDG - summary

Original Question

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Hello dear flowers,
for those interested in LacZ detection by FACS using the FDG substrate I 
have listed the answers I have got so far. We didn't repeat the experiment 
yet with my collegue who has the Rosa mice but we are planning to use 
osmotic shock at 37 degrees for 30 min with FDG + Verapamil (to reduce the 
leaking of the FDG out of the cells) and Chloroquine (to reduce degradation 
of the FDG by the lysosomes) as this protocol seems to work the best for 
the loading of the cells rather than the MP kit. X-gal activity has been 
proven in our mice by histology.
I will keep you updated when we will have the results.
Greetings,
Simona


Simona ZOMPI
MD,PhD
Hubrecht Laboratory
Uppsalalaan, 8
3584 CT Utrecht
The Netherlands
Tel: +31 (0)30 212 1846
Fax: +31 (0)30 212 1801


Support Care International
http://www.care.org


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Responses

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1/Dear Simona,

I suppose this excerpt from the Molecular Probes handbook will explain it:

The fluorescent hydrolysis product of FDG is fluorescein 
(<http://www.invitrogen.com/content.cfm?pageid=8012&sku=F1300>F1300, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=F36915>F36915; 
<http://probes.invitrogen.com/handbook/sections/1001.html>Section 10.1; 
<http://probes.invitrogen.com/handbook/figures/1042.html>Figure 15.3), 
which rapidly leaks from cells under physiological conditions, making the 
use of FDG problematic for prolonged studies.

Below you can see how fast the leakage is. It doesn’t matter if the 
substrate is FDG or FDA.


Figure 15.3 Loading and retention characteristics of intracellular marker 
dyes. Cells of a human lymphoid line (GePa) were loaded with the following 
cell-permeant acetoxymethyl ester (AM) or acetate derivatives of 
fluorescein: 1) calcein AM 
(<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C1430>C1430, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C3099>C3099, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C3100MP>C3100MP), 2) 
BCECF AM 
(<http://www.invitrogen.com/content.cfm?pageid=8012&sku=B1150>B1150), 3) 
fluorescein diacetate (FDA, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=F1303>F1303), 4) 
carboxyfluorescein diacetate (CFDA, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C1354>C1354) and 5) 
CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C2925>C2925, 
<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C7025>C7025). Cells 
were incubated in 4 µM staining solutions in Dulbecco's modified eagle 
medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation 
for 30 minutes, cell samples were immediately analyzed by flow cytometry to 
determine the average fluorescence per cell at time zero (0 hours). 
Retained cell samples were subsequently washed twice by centrifugation, 
resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by 
flow cytometry. The decrease in the average fluorescence intensity per cell 
in these samples relative to the time zero samples indicates the extent of 
intracellular dye leakage during the 2-hour incubation period.


Thus, I would recommend to analyse the samples as soon as possible or to 
switch to another substrate. If you would like to continue in using FDG, 
there are cheaper sources.

Hope this is a bit of help.

Best regards,
Arne
a.schulz@mobitec.de

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2/Dear Simona,

Did you play with conditions for osmotic shock, perhaps substrate doesn't 
get in? Also, can you proof bioactivity with X-gal staining?
Beware however, passive transfer of enzyme from cell to cell can occur 
during osmotic shock (in my hands it always did, so I turned to GFP from 
then on).
Veel geluk,

Bruno Verhasselt

Prof. dr. B. Verhasselt
Ghent University Hospital
Dpt. of Clinical Chemistry, Microbiology and Immunology
2P8 UZ Gent
De Pintelaan 185
B-9000 Gent
Tel +32-92402226
Fax +32-92404985
<mailto:Bruno.Verhasselt@UGent.be>Bruno.Verhasselt@UGent.be


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3/ Hi Simona,

we also want to do that experiment with spleenocytes of Rosa mice in the 
near future. If you get any useful comments, please share them with us.

Some years ago, I did some FDG staining (MP) with thymocytes of lacZ knock 
in mice. The results were reproducible. As far as I can remember one of the 
critical steps was the loading of the dye at the appropriate temperature. 
So we prepared buffers at the right temperature for quick and easy transfer 
of the cells. But I was in the lucky situation to have a lacZ positive cell 
line as positive control, which is no longer available to me.

Thanks in advance

Steffen
 stschmit@uni-mainz.de 



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4/ Hi maybe you might try indirect intracellular staining: stain your cells
of interest with anti-LacZ(1st Ab)  then use 2nd antibody conjugated
FITC or PE  but also against the first Ab. By this way you can also run
FLOW to see if any LacZ positive cells are there.

Is  it possible to get the LacZ excited by laser? If not it might
explain why FACS fails.

Li
chen-22@medctr.osu.edu
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5/ Perhaps the substrate in not getting into your cells. M Probes sells 
more than one kit to help evaluate substrate loading (Detectagene vs Imagene).
One uses hypotonic and one uses lipophilic loading.

We've also had some luck with fixing the cells and staining with anti-LacZ 
antibodies.


---Dennis
le FDG (Sigma F-2756, dilue en DMSO) à 200 µM, le R-(+)-Verapamil
(sigma V-106 dilue en H2O) à 200 µM, et la Chloroquine diphosphate (Sigma
C-6628 dilue en H2O) à 200 µM
djyoung@ucsd.edu 
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6/ Marcos
le FDG (Sigma F-2756, dilue en DMSO) 200 µM, R-(+)-Verapamil
(sigma V-106 dilue en H2O) 200 µM, Chloroquine diphosphate (Sigma
C-6628 dilue en H2O) à 200 µM
Osmotic shock at 37 degrees