Modified from ‘Rat Alveolar epithelial cell isolation and primary culture’ by K. Ritchie, L. Kandalaft, L. Campbell and M. Sakagami.  2003. Pharmaceutical Cell Biology Group, School of Pharmacy, Cardiff University

10X Solution (500 mL)  (Could try substituting serum-free Medium)

• 38.86 g NaCl
• 1.94 g KCl
• 1.42 g Na2HPO4.2H2O
• 0.78 g NaH2PO4.2H2O
• 11.92 g HEPES
• 5.00 g Glucose

Dissolve in 450 mL of distilled water, adjust pH to 7.4 with 5 M NaOH.  Adjust volume to 500 mL.  Filter sterilize, aliquot in 50 mL tubes and store in the dark at 4oC.

• Stock Dnase I (type V)
• 8500 U of Dnase I is dissolved 11 mL of  1X Solution (diluted from 10X solution) with vigorous shaking.  Filter sterilize
• Low ConcentrationDnase I (type V)
• 4.5 mL of Stock Dnase I
• 35 mL of 1X Solution (diluted from 10X Solution)

Prepare Percoll on day of use in TC hood and store on ice.

Light Percoll                                                            Heavy Percoll

6.8 mL of Percoll                                                16.225 mL Percoll

125 mL of FBS                                                125 mL of FBS           

15.7 mL of phenol red-colored water                   6.275 mL water

2.5 mL of 10X solution                                   2.500 mL of 10X solution

Prepare 2 50 mL tubes with 10 mL of Heavy Percoll in the bottom and 10 mL of Light Percoll on layered top of the heavy Percoll. 

Layer about 15 mL of cells on top ot the light Percoll (Most likely prior to antibody.  The rat protocol does this on crude cell suspensions).

Centrifuge at 250 x g, 6oC for 20 min.  Maximum acceleration, no brake.  Debris stays in the light Percoll.  Cells collect at the interface of the Light Percoll (red) and Heavy Percoll.   After  centrifugation, remove the cell debris layer. 

Collect the cell layer and place in a 50 mL tube containing 35 mL of low conentration Dnase.  Centrifuge at 250 x g, 6oC, 20 min. with maximum acceleration and deceleration. 

Remove the supernatant. 

Add Solution needed for the next step (either FC or antibody incubation) and resuspend cells.