A standardized method to study immune responses using porcine whole blood
- Author:
Sameer-ul-Salam MATTOO
1
;
Ram Prasad AGANJA
;
Seung-Chai KIM
;
Chang-Gi JEONG
;
Salik NAZKI
;
Amina KHATUN
;
Won-Il KIM
;
Sang-Myeong LEE
Author Information
- Publication Type:Original Article
- From:Journal of Veterinary Science 2023;24(1):e11-
- CountryRepublic of Korea
- Language:English
-
Abstract:
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.
Objectives: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.
