Mithras luminometer

Env-pseudotyped virus (PSV) 93MW965.26 and infectious molecular clone (IMC) ConM¹⁵ (link) were produced in HEK293T cells, tittered and used in TZM-bl assay to determine nAb responses as previously described.⁵³ (link) Briefly, duplicates of six steps of 3-fold dilution, starting with 1:20 of each serum, were incubated with viral supernatant (at relative luminescence units (RLU) between 150,000 and 200,000) for 1 h. Thereafter, 10⁴ TZM-bl cells were added, and plates incubated for 48 h at 37°C, when Bright-Glo Luciferase assay system (Promega, Madison, Wisconsin, USA) was added to measure luciferase activity with a Mithras luminometer (Berthold, Germany). Positive controls were sera of HIV-1-infected individuals and monoclonal antibodies with known neutralizing titers. Testing against VSV was used to exclude unspecific reactions. Neutralization titers were defined as the sample dilution at which RLU were reduced by 50% compared to virus control wells after subtraction of background RLU in control wells with only cells. Inhibitory concentrations 50 (IC50) were calculated with a linear interpolation method using the mean of the duplicate responses.⁵³ (link)

pRL-Con contains the humanized Renilla Luciferase coding region. pRL-3xBulge-miR122 contains three miR-122 binding sites downstream of Renilla Luciferase (RL) coding region. pGL3FF (Promega) encodes a Firefly Luciferase (FL) gene under the SV40 promoter. The RL-Con/RL-3xBulge-miR122 was cotransfected with FL plasmids in 6-well plates, as described earlier [58 (link)]. The cells were then split after 24 h into plates with thin coatings of Collagen and Matrigel (with the lowest concentrations to avoid cell lysis problems) or into Control plates. The cells were then allowed to grow for 72 h before the relative activities were measured using a Dual-Luciferase Assay Kit (Promega, Madison, WI) following the supplier’s protocol on a Mithras Luminometer (Berthold Technologies). Two independent experiments with two technical replicates were assayed for each condition (with individual Controls).

HEK293 cells were seeded into 24‐well plates at 20,000 cells/well, and incubated at 37°C for 24 hrs. Luciferase constructs (with ~1‐kb sST2 3′‐UTR mRNA) and micro RNA (miRNA) mimics were cotransfected using lipofectamine 2000 (Life Technologies), following the manufacturer's instructions. At 48 hrs after transfection, cells were lysed and luciferase assays performed with the Dual‐Luciferase Reporter Assay System (Promega) on a single automatic injection Mithras luminometer (Berthold Technologies, Bad Wildbad, Germany), following the manufacturer's instructions. Ratios of firefly luciferase to Renilla luciferase readings were calculated.

The method for measuring the chitin-induced ROS burst levels is described in [54 (link)]. Briefly, leaf discs were cut from 7-day-old rice seedlings and floated on water overnight, and then the water was removed. After that, the leaf discs were incubated with 100 µL reaction solution (20 µM luminal, Sigma Aldrich, Saint Louis, MO, USA, Cat. No. 123072 and 2.5 µg/mL peroxidase, Solarbio Science & Technology Co., Ltd. Beijing, China, Cat. No. P8020) containing 400 nM chitin (Sigma-Aldrich, Saint Louis, MO, USA, Cat. No. H1396) to immediately detect the ROS burst. The luminescence was measured by a Berthold Mithras luminometer (Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany) every 1~2 min for 55 min. Eight replicates were performed for each sample.

Leaf discs from 7-day-old rice seedlings were floated on sterilized water overnight, then put into 100 ul reaction solution (20 uM luminal and 2.5 μg/mL peroxidase) containing 500 nM flg22 or 400 nM chitin to immediately test the ROS burst. The luminescence was measured by Berthold Mithras luminometer every 1–2 min for 1 h. Each data point represented eight replicates.