Journal: STAR Protocols

Article Title: Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

doi: 10.1016/j.xpro.2022.101321

Figure Lengend Snippet:

Article Snippet: DNA Clean & Concentrator-25 (Capped) , Zymo Research , Cat# D4033.

Techniques: Recombinant, Electroporation, Modification, SYBR Green Assay, Transfection, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, DNA Extraction, Software, Real-time Polymerase Chain Reaction, Protein Purification, Plasmid Preparation, Synthesized, Flow Cytometry, Spectrophotometry

Journal: Respiratory Research

Article Title: Smoking decreases the response of human lung macrophages to double-stranded RNA by reducing TLR3 expression

doi: 10.1186/1465-9921-14-33

Figure Lengend Snippet: AMø of current smokers show reduced expression of TLR3 mRNA transcripts and intracellular protein relative to AMø of never-smokers. A . mRNA transcripts. RNA was isolated from AMø, depleted of contaminating genomic DNA, reverse-transcribed and analyzed by quantitative real-time RT-PCR using Taqman chemistry and specific primer-probe sets, normalized to GAPDH transcripts. Data are expressed on the horizontal axis as mean ± SEM for relative quantity (dRn), calculated in comparison to a single never-smoker who was arbitrarily designated the reference sample. Never-smokers ( n = 6), red bars; smokers ( n = 11), blue bars. *, p < 0.05 by Mann–Whitney test. B . Intracellular TLR3 (top panel) and TLR9 (bottom panel) expression by flow cytometry. AMø were permeabilized, stained for TLR3 and TLR9 expression and analyzed by flow cytometry. Data are expressed as TLR-positive AMø as mean ± SEM, never-smokers ( n = 10 for TLR3, n = 5 for TLR9), red circles; smokers ( n = 13 for TLR3, n = 6 for TLR9), blue circles. *, p < 0.05 by Mann–Whitney test. C . Immunohistochemical staining. Cytospins from a representative smoker (right) and a never-smoker (left), stained for intracellular TLR3 expression using AEC (red product) and hematoxylin (original 20 X). Top row, isotype control staining, bottom row, specific TLR3 staining. Representative of three experiments with similar results.

Article Snippet: We used antibodies against the following antigens (clones in parentheses): CD45 (2D1) (eBioscience, San Diego, CA), TLR3 (40C1285.6), TLR7 (polyclonal), TLR9 (26C593.2) (Imgenex Corp., San Diego, CA), and TLR8 (303F1.14) (Dendritics, Lyon, France).

Techniques: Expressing, Isolation, Reverse Transcription, Quantitative RT-PCR, Comparison, MANN-WHITNEY, Flow Cytometry, Staining, Immunohistochemical staining, Control

Journal: Respiratory Research

Article Title: Smoking decreases the response of human lung macrophages to double-stranded RNA by reducing TLR3 expression

doi: 10.1186/1465-9921-14-33

Figure Lengend Snippet: Exposure to CSE specifically and rapidly reduces TLR3 gene expression in a differentiated human Mø cell line. THP-1 cells differentiated using PMA & vitamin D3 for 48 hours were cultured in complete medium alone or with various concentration of CSE for an additional 4 hr, then analyzed by quantitative real-time RT-PCR. Data are mean ± SEM of four independent experiments, expressed as dRn (relative to differentiated THP-1 cells cultures in complete medium alone). A , TLR3; B , TLR7; C , TLR8; D , TLR9; E , RIG-1; F , MDA-5; G , PKR. Kruskal-Wallis one-way ANOVA was used to compare groups. *, p < 0.05, compared to cells that did not receive CSE exposure.

Article Snippet: We used antibodies against the following antigens (clones in parentheses): CD45 (2D1) (eBioscience, San Diego, CA), TLR3 (40C1285.6), TLR7 (polyclonal), TLR9 (26C593.2) (Imgenex Corp., San Diego, CA), and TLR8 (303F1.14) (Dendritics, Lyon, France).

Techniques: Gene Expression, Cell Culture, Concentration Assay, Quantitative RT-PCR