Supplementary Materialsijms-21-04180-s001. the specific protein was eluted with 1x PBS containing 500 mM imidazole. The correct identity of the eluted protein was confirmed by running it on a 15% SDS-PAGE gel under reducing conditions, stained with Coomassie blue and the protein band at approximately 20 kDa was subjected to MALDICTOF/TOF MS analysis as previously described . 4.2. Production of mAb (3C8) Specific to CD4-2 Lymphocytes from Olive Flounder The purified recombinant CD4-2 antigen was used to immunize three 6-week-old female BALB/c mice. The antigen (150 g) was mixed with Freunds complete adjuvant (FCA) (1:1 for 3 min. Prior to incubation with mAb, cells were blocked with 0.1% bovine serum albumin (BSA) in 1 PBS for 30 min. Leukocytes were then treated with mAb 3C8, followed by FITC-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pennsylvania, USA) for 1 h. Two-color flow cytometry analysis was conducted against cell surfaces between CD3 and CD4-1, between CD3 and CD4-2, and between CD4-1 and CD4-2. For the analysis of cell surface between CD3 and CD4-1, leukocytes from tissues were first incubated with mAb 10F8 (anti-flounder CD4-1 mouse IgG2) followed by PE-conjugated goat anti-mouse IgG2 for 1 h. Leukocytes were washed with 1 PBS 3 times and then reacted with mAb 4B2 (anti-flounder CD3 mouse IgG1) followed by FITC-conjugated AffiniPure goat anti-mouse IgG1. The analysis MMP7 of cell surface area CD4-2 and CD3 was performed identical way. Leukocytes had been stained with mAb 3C8 (anti-flounder Compact disc4-2 mouse IgG2b) accompanied by PE-conjugated goat anti-mouse IgG2. After cleaning, leukocytes had been treated with mAb 4B2 accompanied by FITC-conjugated AffiniPure goat anti-mouse IgG1. For two times staining with Compact disc4-2 and Compact disc4-1, leukocytes had been 1st incubated with mAb 3C8 after biotinylated with NHS 648 ester (BioActs, Incheon, Korea) that could show reddish colored fluorescence. Cells had been washed and reacted with mAb 10F8 accompanied by FITC-conjugated AffiniPure goat anti-mouse IgG2. Cells binding with mAbs had been analyzed with a FACSCaliburTM (BD biosciences, Bedford, Massachusetts, USA). At least 30,000 occasions had been measured for every test. 4.7. Immunofluorescence Staining The Compact disc4-2-positive HEK 293F Vilazodone Hydrochloride cells had been set onto 8-well chamber slides with 4% paraformaldehyde (Intron, Sungnam, Korea) for 15 min. Your final concentration of just one 1 105 cells through the head-kidney had been prepared on the slide glass utilizing a cytological centrifuge (Hanil Technology Industrial, Gimpo, Korea) at 30 for 5 min. After centrifugation, the cells had been set with 4% paraformaldehyde for 15 min, clogged with 0.1% BSA in 1 PBS for 30 min, and stained with anti-CD4-2 mAb (3C8) for 1 h, accompanied by FITC-conjugated AffiniPure goat anti-mouse IgG for 1 h. Adverse controls had been just stained with FITC, and three washes with 1 PBS had been completed between each stage. Cells had been after that stained with DAPI for 10 min at room temperature. HEK293F cells and leukocytes recognized by mAb (3C8) were examined under a fluorescence microscope, Olympus FV 1000 (Olympus, Seoul, Korea). 4.8. RT-PCR with Flow Cytometry Sorted Leukocytes Leukocytes (1 106 cells/mL in 1 PBS) from the spleen and head-kidney were prepared and stained as Vilazodone Hydrochloride described in the flow cytometry section and sorted using a FACSARIA III cell sorter (BD Biosciences, San Jose, USA). Lymphocytes from the spleen and head-kidney were separated into two groups: 3C8-positive and -negative cells. Total RNA was extracted from 30,000 sorted cells of each population using an easy-BLUE Total RNA Extraction Kit (Intron, Sungnam, Korea) and reverse transcribed into cDNA using a TOPscript cDNA Synthesis Kit with Oligo (dT) primers (Enzynomics, Daejeon, Korea) according to the manufacturers instructions. Specific primers, including CD3, CD4-1, CD4-2, CD8, CD8, TCR, TCR, IgL, IgM and -actin were used for the RT-PCR and are shown in Table 1. For the RT-PCR, 1 L of cDNA template and 10 pM of each primer were used together with an Vilazodone Hydrochloride AccuPower ProFi Taq PCR premix (Bioneer, Daejeon, Korea). The PCR conditions were as follows: one cycle of 95 C for 3 min, 34C40 cycles at 95 C for 20 s, 55C65 C as the annealing.