Fitc anti rat cd4

After stressed 1d, 3d, 5d and 7d, 100 μl samples of anti-coagulated whole blood were collected. The whole blood was then stained with FITC anti-rat CD4 (#201505, Biolegend, USA) and PE anti-rat CD8a (#200608, Biolegend, USA) for 15–20 min, while shielded from light at room temperature of 22 ± 2°C. After incubation, the samples were fixed with 2ml 1X red blood cell (RBC) lysis buffer (#420301, Biolegend, USA) for 10 min, while shielded from light at room temperature, before being centrifuged at 350g for 5 min. The resulting supernatant was discarded, and cells were washed and resuspended in at least 2 ml of cell-staining buffer for flow cytometry analysis (Beckman CytoFLEX, USA).

Anticoagulated blood (100 μl) and treated blood (100 μl) from each sample were pipetted to the bottom a flow-cytometry sample tube and appropriate amounts of FITC anti-Rat CD4, PE anti-Rat CD8, APC anti-Rat CD3, PE anti-Rat IL-4, and APC anti-Rat IFN-r antibodies (BioLegend, San Diego, CA) were added and mixed. Then samples were incubated for 20 min at room temperature in the dark. Two microliters of 1 × red cell lysate were add to the samples, mixed well, and centrifuged 5 min at 300 ×g to collect supernatant, which was added to 2 ml cell washing buffer, mixed well, and centrifuged 5 min at 300 ×g to collect supernatant. Subsequently, 500 μl cell washing buffer was add to the supernatant and mixed well before flow cytometry (BD LSRII Flow Cytometry Instrument, BD Corporation).

Amylase, lipase, D-lactate, soluble tumor necrosis factor-α receptor (sTNF-αR), and secretory immunoglobulin A (sIgA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). The chromogenic limulus amebocyte lysate kit was purchased from Bioendo Technology Co., Ltd (Xiamen, China). Rat interleukin-1beta (IL-1β) and transforming growth factor-β (TGF-β) ELISA kits were purchased from ExCell Biotech Co., Ltd (Shanghai, China). Rat TNF-α and IL-10 ELISA kits were purchased from Cusabio Biotech Co., Ltd (Wuhan, China). Rat IL-6 and IL-18 ELISA kits were purchased from Elabscience Biotech Co., Ltd (Wuhan, China). The rat IL-8 ELISA kit was purchased from Neobioscience Technology Co., Ltd (Shenzhen, China). The rat IL-4 ELISA kit was purchased from Invitrogen (Carlsbad, CA, USA). The specific antibodies against FITC anti-rat CD4, Alexa fluor^®647, anti-rat IFN-γ, PE anti-rat IL-4, and Alexa fluor^® 647 anti-rat Foxp3 were purchased from BioLegend (San Diego, CA, USA). Rabbit anti-rat CD68 and rabbit anti-rat CD103 were purchased from Abcam (Cambridge, MA, UK). PE anti-rat CD25 was purchased from BD Biosciences (San Jose, CA, USA). The sources of other reagents were indicated in the specified methods.