Protocol for loading human ALIs with Fluo-4-AM (3-26-12, modified 9-24-18)
1.) Wash ALI cultures to remove media from basolateral side (~ 4 times with 500 µl HBSS + glucose). It is important to remove all traces of serum for loading. Serum contains esterases that inhibit loading. Put 600 µl HBSS on basolateral side.
2.) Wash apical side to remove mucus/debris/media (~ 4 times with 200 µl HBSS w/o glucose). Aspirate solution and leave dry.
3.) Make a working solution of Fluo-4. Add to a 1.5 ml eppendorf (in order indicated)
a. 1 ml HBSS w/o glucose
b. 4 µlProbenecid stock solution (vortex after addition)
c. 2 µl Pluronic stock solution (vortex)
d. 10 µl Fluo-4 stock solution (vortex)
4.) Add working Fluo-4 solution to the apical side of the ALIs (~50 – 100 µl per ALI); basolateral side stays in HBSS + glucose, no Fluo-4.
5.) Incubate at room temp for ~ 90 min to 2 hrs.
6.) Wash apical side of cultures 3 times with HBSS w/o glucose
7.) Let cultures incubate for 15 min at room temp before use (to allow for de-esterification/dye retention) with 30 uL HBSS w/o glucose on top
8.) Image as desired using Fluo-4 settings on the confocal (488 nm laser) or FITC/GFP filter set.
Stock Solutions
Probenecid (250x)
· Dissolve 75 mg probenecid (Sigma P8761) into 500 µl 1 M NaOH
· Vortex to dissolve; incubate 5 min at room temp to ensure that all powder is dissolved
· Add 500 µl of 1 M HEPES or 10x PBS (final volume = ~1 ml)
· Store at room temp
· Make a fresh solution every week or if you see a precipitate form
20% Pluronic F127 (500x)
· Dissolve 0.2 g in 1 ml DMSO; heat to 37°C and vortex to dissolve
· Store at room temp
· Make a fresh solution every week or if you see a thick cloudy precipitate form
Fluo-4-AM (Invitrogen F-14201)
· 1 tube (50 ug) of Fluo-4 + 46 µl DMSO = 100 x solution
· Aliquot 5 or 10 µl aliquots into PCR tubes and freeze at -20°C
