Reaktion #520223

Reaktionsgleichung

Fluorescein diacetate

→

fluorescein

Lösungsmittel

dimethyl sulfoxide

Reaktionsbedingungen

Aufarbeitung

1. 1
   Sonstigeto produce a 1 mg/ml stock solution
2. 2
   FiltrationThe stock was filtered through 0.8 μm nylon
3. 3
   SonstigeFor individual assays, an intermediate stock was prepared by thawing
4. 4
   workup.ADDITIONdiluting an aliquot with RPMI-1640+10% Fetal Calf Serum (FCS) such that
5. 5
   Sonstigewould produce a final concentration of 8 μg/ml
6. 6
   Sonstigereaction with endogenous enzymes in the FCS (which
7. 7
   Temperaturcould increase the background fluorescence

Vorschrift

Fluorescein diacetate (FDA) (Sigma Chemical Co., St. Louis, Mo.), or 2′,7′-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes, Inc., Eugene, Oreg.) was dissolved in dimethyl sulfoxide (DMSO) to produce a 1 mg/ml stock solution. The stock was filtered through 0.8 μm nylon, aliquoted into 1.5 ml microfuge tubes and stored frozen at −20° C. in the dark. For individual assays, an intermediate stock was prepared by thawing and diluting an aliquot with RPMI-1640+10% Fetal Calf Serum (FCS) such that adding 50 μl of dye solution to a well would produce a final concentration of 8 μg/ml. To minimize reaction with endogenous enzymes in the FCS (which could increase the background fluorescence by cleaving ester groups to form free fluorescein), intermediate dilutions were prepared immediately prior to loading each individual plate.

Quelle