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        Phagocytosis

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        1224

        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 -20 ° C.

         

         

        For labeling, resuspend the pellet of 2 x 1010 particles in 20 ml Na2 HPO4 (50 mM, pH 9.2)

         

         

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

         

         

         Wash 2 x in Na2 HPO4 (50 mM, pH 9.2) and 4 x in PB.

         

         

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

         

        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 m l cells (2 x 106 with 12 m l 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 m l  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 m l cells and 12 μl unlabeled yeast in 1 ml PB + Trypan blue.

         

         

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

         

         

         Standard curve:

         

         

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

         

         

         

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