Loading of Primary ALIs for Calcium

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