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Johns Hopkins University School of Medicine
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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 μl unlabeled yeast in 1 ml PB + Trypan blue.

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

 Standard curve:

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

 

This page was last edited 06/26/2003