User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/02/11

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CFDA-SE Rotifer Staining
- A. vaga from sub'd 12/16/09 plate 1 well 5 - ~10 rotifers per treament

TREATMENT Test 40ul of CFDA-SE 1) 1:400 dye dilution  a)1 hour b)2 hours 2) 1:800 dye dilution a)1 hour 3) 1:1600 dye dilution a)1 hour

CFDA-SE staining of A. vaga

(1) Transfer 3 ml of spring water (pH 4.5-5) to a glass vessel (small glass petri dish or culture tube).

(2) Add 10 µl of 10 mM CFDA-SE stock. Mix thoroughly. (To make 10 mM CFDA-SE stock, resuspend an aliquot containing 500 µg CFDA-SE in 90 µl ultrapure DMSO. Store frozen at –20oC.))

(3) Add healthy, well fed animals to be stained in 40ul total volume (a 1:400 final dilution of dye; 25 µM final concentration)

(4) Incubate at room temperature for 1-2 hours in a dark location.

(5) Pipette off media in the staining dish and add 200ul filtered spring water. Repeat rinse 3-5 times. Rinsing in the dish with retain most of the adults while eggs and juveniles which do not attach as well may be lost. To retain younger animals or if staining in a glass tube: transfer stained animals to a centrifuge tube; spin to pellet all animals; discard supernatant, taking care to not disrupt the animal pellet; add filtered spring water. Repeat 3-5 times.

(6) Incubate the stained animals for 15 minutes at room temperature in 200ul filtered spring water.

(7) Visualize in the GFP channel of a fluorescent microscope.

Notes…
 * CFDA-SE from Invitrogen. Cat no. V12883.
 * Filtered Poland Springs spring water has a pH between 4.5-5.
 * CFDA-SE passively diffuses into cells. For cultured cells, 15 minutes is sufficient staining time.  For long term persistent staining in A. vaga, the 60 min. staining is necessary.
 * The final 15 minute room temperature incubation is to allow CFDA-SE time to be cleaved by intercellular esterases. This produces a highly fluorescent compound which will not diffuse out of the cell/animal.
 * Stained this way, the fluorescent dye persists in animals in excess of three weeks. It stains the mastax most strongly and also highlights a sac like structure at the posterior end of the animal, diffusely stains the head region and the gut.  Other than the mastax, other stained structures will not always maintain high intensity fluorescence for ≥ 21 days.



1:400 dilution stained 2 hours - rotifer died at 2:15pm



1:800 dilution stained 1 hour - rotifer alive after 20 hours

Arrow = stained Mastex

1:1600 dilution too faint to distinguish mastex