Chlorophenol red β d galactopyranoside cprg

Renilla and firefly luciferase activities were determined using the Dual-Luciferase^® Reporter Assay System (Promega) according to the manufacturer’s instructions. IRES activity was determined from the ratio of firefly to Renilla luciferase activity (Thermo/Fluoroskan Ascent™ FL) according to the manufacturer’s instructions. β-galactosidase activities were determined using the chlorophenol red-β-D-galactopyranoside (CPRG) (Sigma-Aldrich) according to the manufacturer’s instructions. β-galactosidase value were determined the absorbance at 570 nm.

Wild-type (WT) C57BL/6 and BALB/c mice were purchased from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). TLR2 knockout (KO), TLR4 KO, MyD88 KO and OT-I mice were purchased from the Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). PD-L1 KO mice were purchased from The Jackson Laboratory (MMRRC stock # 32234, Bar Harbor, USA). All animal experiments were approved by the Animal Care and Use Committee of Southeast University.
4T1, B16F10, EL4 and Hepa 1–6 were cultured in RPMI 1640 (HyClone) supplemented with 10% FBS (Gibco), 0.05 mM 2-ME (Gibco) and 50 μg/ml Gentamicin (Lonza). Raw264.7 were cultured in complete DMEM (HyClone) medium. B16F10 tumor cells stably transduced with Beclin1-specific short hairpin RNA (shRNA) (BECN1-KD B16F10) and scrambled shRNA (Ctrl-B16F10) were generated previously. All cell lines were detected for Mycoplasma every 2 weeks and were negative prior to use.
LPS, Chloroquine diphosphate, Chlorophenol red-β-D-galactopyranoside (CPRG), and Proteinase K were purchased from Sigma (St. Louis, USA). SB203580 and Stattic were purchased from MCE (Shanghai, China). Recombinant Murine M-CSF, IFN-γ and IL-4 were purchased from PeproTech (Rocky Hill, USA). CFSE was purchased from Invitrogen.

In vitro assays against T. cruzi were performed as described previously [12 (link)]. HFF-1 human fibroblasts were seeded at 4x10³/well in 96-well tissue culture plates in DMEM (Cultilab) without phenol red and then incubated overnight (37°C, 5% CO₂). Next, trypomastigotes were added at 4x10⁴/well, and the plates were incubated (37°C, 5% CO₂). After 24 h, 2-fold serial dilutions of the test compounds were added at concentrations ranging from 64 to 0.12 μM, and the plates were then incubated (37°C, 5% CO₂). Each compound concentration was evaluated in duplicate. All the plates included BZ (64–0.12 μM) (Sigma-Aldrich) as a positive control and untreated wells (100% growth) as a negative control. After 120 h, chlorophenol red β-D-galactopyranoside (CPRG, Sigma-Aldrich) was added to each well. The absorbance was measured at 570 nm in an automated SpectraMax 384 microplate reader (Sunnyvale, CA, USA), and the data were analysed using GraphPad Prism version 8.0 (San Diego, CA, USA) for IC₅₀ value calculation.

The drug susceptibility of HIV-1 was determined by an enzymatic assay of β-galactosidase activity using TZM-bl cells expressing CD4 and coreceptors CXCR4 and CCR5. For this, the TZM-bl cells were seeded in 96-well plates (10⁴/well) the day before the experiment. Then, various concentrations of PLA2s were added to the cells, followed by inoculation with HIV-1 pseudoviruses at 500 blue cell-forming units (BFU)/well. After 48 h of inoculation with HIV-1, the cells were lysed with PBS containing 1% Triton-X100 and incubated at 37 °C for 1 h with 10-mM chlorophenol red-β-D-galactopyranoside (CPRG; Sigma, St. Louis, MO, USA) in 2-mM MgCl₂ and 100-mM KH₂PO₄. The reaction was stopped by adding 0.5-M Na₂CO₃. Optical density at 570 nM was measured on a microplate reader (Hidex Sense Beta Plus, Hidex, Turku, Finland). Drug concentrations that brought about 50% inhibition of the β-galactosidase activity were determined.