- 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.
- 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.
- Make working solution of DAF-FM diacetate (DAF-FM-DA). Add to a 1.5 ml eppendorf tube (in order indicated)
- 1 ml glucose-free HBSS or PBS
- 5 µL cPTIO stock solution (recipe below)
- 10 µL DAF-FM-DA stock solution (recipe below)
- Add working DAF-FM solution to the apical side of cultures (~50 µL per 0.33 cm2 transwell).
- 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.
- After 90 min, wash apical side of cultures 3 times with 100-200 µL glucose-free HBSS or PBS.
- Incubate cultures 10-15 min at room temp before use to allow de-esterificaiton and dye retention.
- Image as desired using Fluo-4, FITC, AF488 settings (your microscope may vary).
- 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)
(RJL 5-16-12)