Implementation of the EGFP-K562 flow cytometric NK test: determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding.
BACKGROUND
Natural Killer (NK) cells are key actors of innate immunity that supervise the organism's cells, and fight against viral infections and cancer development through their cytotoxic activity. This cytotoxic activity is modulated by cytokines and hormones and could be influenced by physiological or pathological conditions. New techniques for measuring NK cytotoxic activity by flow-cytometry have recently been developed, and they correlated strongly with the standard chromium ((51)Cr) release assay. Our aim was to implement a previously published enhanced green fluorescent protein (EGFP)-K562 flow cytometric method and use it to evaluate NK cytotoxic activity under different nutritional conditions.


METHODS
NK effector cells were isolated from peripheral blood mononuclear cells, and a K562 cell line stably transfected by EGFP was used as target cells. Different analytical parameters, including cell ratios and incubation times, were studied to improve the EGFP-K562 flow cytometric NK test conditions.


RESULTS
The optimized test was then used to determine the effect of fasting and refeeding on NK cell numbers and activity in a physiological situation. NK cytotoxic activity in fasted conditions (30.4 +/- 4.4%) increased by a factor 1.7 +/- 0.2 (P = 0.0025) in nourished conditions (45.0 +/- 4.6%) in healthy elderly people.


CONCLUSIONS
Therefore, this method provides a reliable, reproducible and rapid test for analyzing NK cytotoxicity under various conditions.
Allegra
Séverine
S
Laboratoire de Biochimie, Biologie Moléculaire et Nutrition EA2416, Faculté de Pharmacie, IFR 79 Santé-Auvergne, CLARA, Université Clermont 1, Clermont-Ferrand, France.
Deleine
Cécile
C
Michael-Jubely
Rime
R
Gryson
Céline
C
Boirie
Yves
Y
Kantakamalakul
Wannee
W
Vasson
Marie-Paule
MP
D000368:Aged; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D003602:Cytotoxicity, Immunologic; D004435:Eating / Q000276:immunology*; D005215:Fasting; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology*; D008297:Male
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins