Lee Lab: DAF-FM

Loading ALIs with DAF-FM

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)

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)

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