Journal: Frontiers in Immunology
Article Title: BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection
Figure Lengend Snippet: BATF silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg GFP Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P
Article Snippet: Per 5 × 106 PBMCs from ATB patients were resuspended in 100 μL room-temperature balanced Nucleofector®solution (Human T Cell Nucleofector®Kit, Lonza, Germany), and added with 200 nM siRNA (ON-TARGET plus Non-targeting pool or BATF ON-TARGET plus SMART pool, GE Dharmacon, USA) or 2 μg GFP Vector (Lonza, Germany).
Techniques: Fluorescence, Microscopy, Transfection, Electroporation, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Cell Culture, Small Interfering RNA