%0 Journal Article
%T A standardized method to study immune responses using porcine whole blood.
%A Mattoo SU
%A Aganja RP
%A Kim SC
%A Jeong CG
%A Nazki S
%A Khatun A
%A Kim WI
%A Lee SM
%J J Vet Sci
%V 24
%N 1
%D Jan 2023
%M 36726276
%F 1.603
%R 10.4142/jvs.22210
%X BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment.
OBJECTIVE: Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses.
METHODS: Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV).
RESULTS: The frequency of IFN-γ+CD3+ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ+CD4-CD8+ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples.
CONCLUSIONS: We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.