Preparation suggestions for ALEXA 488 labeled SOLUBLE FLAER, cat. No. FL2S.
The reagent has been shipped AS A LIQUID in 1.0 ml of buffer at a concentration of 10-6
M. It can be stored at 4 OC in the refrigerator. IT DOES NOT NEED TO BE FROZEN,
BUT as with other fluorescent materials, it should be protected from long exposure to
light.
Liquid FLAER is a new reagent and its shelf life has not been firmly established. The
reagent is intended for research purpose only.
We suggest using the reagent at a final concentration of 5 x 10-8 M (for example, by
diluting 5 ìL into a final volume of 100 ìL containing cells), however you might wish to
try other concentrations for your own application. We recommend that you do not dilute
the reagent before use.
Suggested simple method for using SOLUBLE FLAER to detect GPI-anchored
protein-deficient cells by flow cytometry. See below for methods used in PNH
diagnosis.
1 x 106 cells are washed once in cold phosphate-buffered saline (PBS) by centrifugation
at 1100 rpm at 40C for 10 minutes and resuspended in 250 ìL of cold PBS. FLAER is
added to a final concentration of 10 nmol/L (5 x 10-8M). The mixture is incubated in the
dark on ice for 20 minutes. The cells are then washed and resuspended in cold PBS, and
fixed by adding an equal volume of 2% paraformaldehyde. Analysis of FLAER binding
is performed using a flow cytometer equipped with 488-nm argon ion laser. A sample of
cells known for GPI-anchor expression is stained at the same time and used as a positive
control to discriminate the FLAER negative population in the tests.
There are a number of published methods describing the detection of PNH using
FLAER in combination with labeled antibodies. One example is Sutherland et al. in
Cytometry Part B (Clinical Cytometry) 72B: 167-177 (2007). We can provide a
detailed protocol upon request.
Please contact us if you have any questions.