β galactosidase assay kit

To quantify the fusion promotion ability of HN mutants, a reporter gene method was performed as previously described [19 (link), 20 (link)]. BHK-21 cells were plated in both 12-well plates and 60-mm culture dishes. As the confluency reached 80%, the cell monolayers in the 12-well plates (monolayers A) were infected with recombinant vTF7–3 for an hour and then co-transfected with the desired HN mutants and wt F genes. The cell monolayers in the 60-mm culture dish (monolayers B) were infected with wt vTF7–3 and transfected with plasmid pG1NT7β-gal encoding β-galactosidase. At 16 hpt, equal numbers (1 × 10⁵) of monolayers A and B were mixed in 96-well plates for further incubation. After incubation at 37 °C for 7 h, the β-galactosidase activity of cells was assayed with a β-Galactosidase Assay Kit (Beyotime, China). The level of fusion was quantified by the absorbance at 590 nm with an ELISA reader. The monolayers A co-transfected with the pBSK⁺ empty vector and wt F and co-transfected with wt HN and F were used as negative and positive controls, respectively.

The enzymatic activity of BGLAt was measured by referring to the pNPG method [20 (link)]. Fifty μL of the enzyme solution was mixed with 100 μL of 5 mM p-nitrophenyl-β-D-glucopyranoside, which reacted at 50 °C for 30 min. Then, 1 mL of 1 M Na₂CO₃ was added to terminate the reaction. Absorbance data were obtained using a microplate spectrophotometer at 400 nm. One unit of enzymatic activity was defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per min under assay conditions. The enzymatic activity of BGALAo was analyzed using a β-galactosidase assay kit (Beyotime, Shanghai, China).

The detection of β-galactosidase activity in E. coli MG4/pKDT17, E. coli pEAL08-2 [30 (link),31 (link)], and E. coli pDSY was described previously. The overnight culture of E. coli MG4/pKDT17, E. coli pEAL08-2, and E. coli pDSY was diluted 1:100 in LB and then incubated with falcarindiol at 37 °C for 8 h. A β-galactosidase Assay Kit (Beyotime, Shanghai, China) was used to quantify β-galactosidase activities.

Y1H assays were performed as previously described (Zhou et al., 2020 (link); Xiang et al., 2021a (link)). The pLacZi-proPME31 and pB42AD-ABI5 constructs were transformed into yeast strain EGY48. Transformants were screened on SD/–Trp–Ura plates and cultured in SD/–Trp–Ura/Gal/Raf/X-Gal (80 μg mL⁻¹) plates for color development. The β-galactosidase activity was detected using a β-Galactosidase Assay Kit (Beyotime, Shanghai, China). The β-galactosidase activity was calculated as previously described (Xiang et al., 2021a (link)). The pLacZi and pB42AD vectors were used as the negative control.

Splenocytes (1×10⁶ cells well^–1) from control or MK ablated mice after 7 days L.m.-OVA infection were re-stimulated for 4 hr in vitro with OVA peptide (10 μM) in the presence of Brefeldin-A (BFA, 10 μg ml^–1). Activated T cells were then analyzed by a flow cytometer.
For MK-induced T cell activation, 3×10⁴ MK subpopulations for each sample were sorted and co-cultured with 100 μg ml^–1 soluble full-length OVA for 24 hr, then co-cultured with 6×10⁴ OT-I CD8⁺ T cells or B3Z T cells (Karttunen et al., 1992 (link)) for 48 hr at 37 °C in a 5% CO₂ incubator as described (Zufferey et al., 2017 (link)). OT-I T cell activation was detected by measuring intracellular IL-2 levels. B3Z T cell activation was detected using β-galactosidase Assay Kit (RG0036, Beyotime). Bone marrow-derived dendritic cells (DCs) were adopted as positive controls for T cell activation assay. To obtain bone marrow-derived DCs, isolated bone marrow cells were cultured in RPMI 1640 with 10 ng ml^–1 of GM-CSF and 10 ng ml^–1 of IL-4 as described (Roney, 2019 ).