Infinite n200 pro

Fluorescence-activated cell sorted peritoneal B1a, B1b, and B2 B cells were loaded onto a QIAshredder spin column (QIAGEN, Hilden, Germany) and homogenized. For RNA isolation from the lysate, the RNeasy Mini Kit (QIAGEN) was used according to the manufacturer’s instructions. RNA concentration was measured by a Tecan Infinite N200 PRO (Tecan Austria GmbH, Gröding, Austria). Identical amounts of RNA (30 ng) were used for cDNA synthesis with the QuantiTect Reverse Transcription Kit (QIAGEN). Real-time RT-PCR for S1P1_1–5 receptor expression was performed with the QuantiTect SYBR Green PCR Kit (QIAGEN) on an ABI Prism 7000 Sequence Detection System (SDS) (Applied Biosystems, Foster City, CA, USA). Analysis was done with the ABI Prism 7000 SDS software v1.1 (Applied Biosystems). Semi-quantitative gene expression was calculated according to the 2^−ΔCt method normalized to β₂ microglobulin. The consistency of the internal control was verified by comparing 2^−ΔΔCt values as described by Schmittgen et al. [44 (link)]. Primers were designed using Primer3 software (version 0.4.0, Whitehead Institute for Biomedical Research, Cambridge, MA, USA) and synthetized by BIOTEZ (Berlin, Germany). Primer sequences are listed in Table 1.

ELISA protocol was adapted from Quinlan et al.^{52 (link)}. Briefly, 100 μL of the wild-type GP in PBS was adsorbed to clear 96-well Maxisorp plates (Nunc) at a concentration of 1 μg/mL and left at 4 °C overnight. Plates were washed with PBS with 0.05% Tween 20 (PBST) and incubated with 100 μL of 2% BSA in PBST for 1 h at room temperature. Plates were washed with PBST then threefold serially diluted of anti-Nipah mAb were added to wells followed by incubation for 2 h. After washing with PBST, 100 μL of secondary antibody, goat anti-human IgG H&L conjugated with HRP (1:10,000, Abcam) were incubated for 1 h. Finally, plates were washed, and incubated with 100 μL of TMB substrate (KPL) for 5 min and quenched with 100 μL of 1 N H₂SO₄. Plates were read using a Tecan Infinite N200 Pro plate reader at 450 nm.