Kinase experiment:

The cells are palced in a petri dish and BCECF present in trichomonads is fixed for electron microsopy. Controns without BCECF loading, without DAB treatment, and without UV illumination are run in parallel. Trichomonads remaine viable during the procedure, except that without BCECF they are dead and disrupted by 4 h. Then the cell are lysed. After lysis, BCECF is added to the lysate to 20 μM, and the lysate is incubated,followed by 3 washes in lysis buffer to remove free dye and resuspension of the pellet in the original volume[1].

BCECF is a duorescent dye with λexcitation of 420~520 nm and λemission of 530 nm.

Fully treated cells show hydrogenosomes with an electron-dense deposit which aggregates to a variable extent.The staining is seen in the interior of hydrogenosomes in some instances. It is also observed by microscopy that the K+/H+ ionophor nigericin does not inhibit hydrogenosomal loading with BCECF[1].

[1]. Scott DA, et al. Analysis of the uptake of the fluorescent marker 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) by hydrogenosomes in Trichomonas vaginalis. Eur J Cell Biol. 1998 Jun;76(2):139-45.