This is a cached page for the URL (http://www.hopkinsmedicine.org/cellbio/devreotes/phagocytosis.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Phagocytosis

 

 

Phagocytosis

Fluorescent labeling of yeast

Resuspend 5 grams of yeast in 50 ml PBS in 100 ml flask

Put flask for 30 minutes in boiling water bath while stirring.

Wash 5 x with PBS and 2 x in PB.

Adjust the concentration to of particles to 109 particles/ml. Can now be frozen at -20C.

For labeling, resuspend the pellet of 2 x 1010 particles in 20 ml Na2HPO4 (50 mM, pH 9.2)

Add 2 mg TRITC, incubate 30 minutes at 37C on a rotary shaker.

 Wash 2 x in Na2HPO4 (50 mM, pH 9.2) and 4 x in PB.

Freeze aliquots of 109 particles/ml at -20C.

Phagocytosis assay

Grow cells for at least 5 generation times in HL5, the density not exceeding 5 x 106 cells/ml

 Harvest 2 x 107 cells, wash 1 x in PB and resuspend in 1 ml PB.

 Mix 100 ml cells (2 x 106 with 12 ml labelled yeast 1.2 x 107) in glass tubes.

 Incubate for 0, 5, 10, 20, 30 minutes.

 Stop with 1 ml cold PB.

 Add 100 ml  Trypan blue (2 mg/ml in 20 mM citrate, 150 mM NaCl, pH 4.5).

 Mix and shake for 5 minutes.

 Spin 3 minutes 500 x g.

 Remove supernatant. Resuspend in 1 ml PB.

 Read in fluorescence spectrofotometer at 544 nm excitation, 574 nm emission.

 Include the following blanks:

 For autofluorescence: 100 ml cells and 12 ml unlabeled yeast in 1 ml PB + Trypan blue.

 For background, unquenched fluorescence, 12 ml labeled yeast in 1 ml PB + Trypan blue.

 Standard curve:

 0,2,5,10 ml labeled yeast in 1 ml PB.

 

 

 

 

 

 

 

 

 
Last modified:12/15/99