Optimal preparation of rhesus macaque blood for cytokine flow cytometric analysis.
BACKGROUND
The rhesus macaque is a common substitute for human subjects in many disease models, including simian immunodeficiency virus, the non-human primate equivalent of the human immunodeficiency virus. Monoclonal antibodies and fluorochromes optimized for use in macaques were included in samples examined for immune responses with the use of intracellular cytokine flow cytometry (CFC).


METHODS
Sample preparation was optimized based on the following comparisons: activation of peripheral blood mononuclear cells (PBMCs) versus whole blood; separation of PBMCs using BD Vacutainer cell preparation tubes versus Ficoll; and activation of samples on the day they were collected versus holding samples overnight.


RESULTS
When activated with the simian immunodeficiency virus type mac239 and Gag peptide mix or with the superantigen Staphylococcal enterotoxin B, separated PBMCs produced greater CD4 and CD8 fluorescence intensities and a larger percentage of CD69+ cytokine-positive cells than did whole blood samples. PBMCs separated by cell preparation tubes produced absolute T-lymphocyte counts equivalent to that with Ficoll separation, and CFC results with both methods were similar. When subjected to overnight shipping conditions, whole blood and PBMCs sometimes showed a reduction in mean fluorescence intensity and percentage of CD69+ cytokine-positive T lymphocytes.


CONCLUSIONS
Due to this reduction in responses, it is preferable to activate samples on the day of blood collection. Samples can be surface stained and frozen in BD FACS Lysing Solution, to be thawed at a later date; this preserves their ability to display a cytokine response. Thus optimal CFC results are achieved by separating macaque PBMCs from whole blood, activating samples on day of collection, and, if necessary, freezing samples after surface staining for future analysis.
Nomura
Laurel E
LE
Biological Research and Development, Immunocytometry Systems, BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, USA. laurel_nomura@bd.com
deHaro
Eileen D
ED
Martin
Louis N
LN
Maecker
Holden T
HT
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D015683:Gene Products, gag / Q000737:chemistry; D007371:Interferon-gamma / Q000378:metabolism; D037181:Lectins, C-Type; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008253:Macaca mulatta; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D016097:Simian Acquired Immunodeficiency Syndrome / Q000097:blood; D015302:Simian Immunodeficiency Virus / Q000737:chemistry*; D013601:T-Lymphocytes / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus; D000099192:CD69 Antigens
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D016207:Cytokines; D004768:Enterotoxins; D015683:Gene Products, gag; D007371:Interferon-gamma; D037181:Lectins, C-Type; D010446:Peptide Fragments; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; D054301:gag Gene Products, Human Immunodeficiency Virus; D000099192:CD69 Antigens; C109809:p2 gag peptide, Human immunodeficiency virus 1; C031818:enterotoxin B, staphylococcal