Loading ALIs with DAF-FM
  1. Wash ALIs to remove media from basolateral side (~3-4 times with 500 µL room-temp HBSS + glucose, MEM-amino acids, and vitamins); place 600 µL on basolateral side.  HBSS recipes can be found here.
  2. Wash apical side to remove mucus/debris/media (~4 times with 100-200 µL glucose-free HBSS OR PBS).  Aspirate final wash and leave dry.
  3. Make working solution of DAF-FM diacetate (DAF-FM-DA).  Add to a 1.5 ml eppendorf tube (in order indicated) 
    1. 1 ml glucose-free HBSS or PBS
    2. 5 µL cPTIO stock solution (recipe below)
    3. 10 µL DAF-FM-DA stock solution (recipe below)  
  4. Add working DAF-FM solution to the apical side of cultures (~50 µL per 0.33 cm2 transwell).
  5. Incubate at room temp for 90 min.  If loading multiple cultures, you can stagger loading 10-15 min (based on experiment length) to ensure each culture gets the same loading time.
  6. After 90 min, wash apical side of cultures 3 times with 100-200 µL glucose-free HBSS or PBS.
  7. Incubate cultures 10-15 min at room temp before use to allow de-esterificaiton and dye retention.
  8. Image as desired using Fluo-4, FITC, AF488 settings (your microscope may vary).
  9. Keep loading and settings exactly the same from experiment-to-experiment to allow comparison of results.

Recipes for Some of the Stock Solutions:

Dulbecco’s PBS  (From pre-mixed powder; Gibco 21600/010 or 10x solution of Ca2+-free PBS);
·       Dissolve 1 packet in ~800 ml distilled water
·       Add 2 ml 500x CaCl2 (stock solution recipe below)
·       Add 2 ml 500x MgCl2 (stock solution recipe below)
·       Adjust pH to 7.2 with HCl
·       Volume solution to 1 L
·       Filter sterilize if desired (recommended)
·       Store at room temp

500x CaCl2
·       Dissolve in water to 900 mM; make 25 or 50 ml at a time;
·       Store at room temp

500x MgCl2
·       Dissolve in water to 750 mM; make 25 or 50 ml at a time;
·       Store at room temp

DAF-FM diacetate (Invitrogen F-14201)
·       1 bottle of DAF-FM + 46 µl DMSO = 100x solution
·       Aliquot 5 or 10 µl aliquots into PCR tubes and freeze at -20°C

Carboxy-PTIO (cPTIO)
·       Stock solution is 1 mM in DMSO (store frozen at -20°C); final working concentration is 5 µM; 

         (RJL 5-16-12)