rf10  (Addgene inc)

Journal: Cells

Article Title: Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines

doi: 10.3390/cells13161310

Figure Lengend Snippet: Cytotoxic effector proteins in LGLL cells. Analysis of internal GrzB and Gnly in NK-92 ( A ), NKL ( B ), KHYG-1 ( C ) and MOTN-1 ( D ). Cells were mildly homogenized with a balch homogenizer and subjected to differential and density centrifugation as described. Whole cell lysate (WCL), enriched organelles (EO), crude lysosomal fraction (CLF), and the cytosol (CYT) were separated together with fractions 1–6 of the density gradient on Bis-Tris NuPAGE gels, transferred to nitrocellulose, and tested for the presence of LAMP-1 and GrzB with respective mab from BD Bioscience and BioLegend and HRP-conjugated anti-mouse IgG antibodies from Cytiva. Gnly was stained with a polyclonal goat anti-Gnly antibody from R&D followed by HRP-conjugated anti-goat IgG antibodies from Abcam.

Article Snippet: For granulysin staining, we also used an unconjugated anti-Gnly mab (clone RF10, MBL International, Woburn, MA, USA) with Alexa-Fluor 555-conjugated goat anti-mouse polyclonal antibody (pab) (Thermo Fisher Scientific) and unconjugated anti-Gnly pab from goat (RD Systems, Minneapolis, MN, USA) with Alexa-Fluor 555-conjugated donkey anti-goat pab, respectively.

Techniques: Centrifugation, Staining

Journal: Cells

Article Title: Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines

doi: 10.3390/cells13161310

Figure Lengend Snippet: Colocalization of intracellular LAMP-1 with GrzB and the 15 kDa (RF10) or 9 kDa (pc) form of Gnly in NK-92 cells. Following fixation and permeabilization, cells were stained with FITC-conjugated anti-LAMP-1 mab. After washing, cells were additionally stained with PE-conjugated anti-LAMP-1 mab (as a positive control for colocalization), PE-conjugated anti-GrzB mab, or with anti-Gnly mab RF10 or a polyclonal anti-Gnly pab (pc) and appropriate Alexa Fluor 555-conjugated secondary antibodies. A total of 10,000 cells were acquired with an ImageStream Mark II imaging flow cytometer. Only focused, single cells were considered for further analyses. ( A ) Histograms display the geometric mean value of the BDS score of respective stainings and the percentage of cells displaying a BDS score >2. ( B ) Representative images of stained cells. Scale bars represent 7 µm.

Article Snippet: For granulysin staining, we also used an unconjugated anti-Gnly mab (clone RF10, MBL International, Woburn, MA, USA) with Alexa-Fluor 555-conjugated goat anti-mouse polyclonal antibody (pab) (Thermo Fisher Scientific) and unconjugated anti-Gnly pab from goat (RD Systems, Minneapolis, MN, USA) with Alexa-Fluor 555-conjugated donkey anti-goat pab, respectively.

Techniques: Staining, Positive Control, Imaging, Flow Cytometry

Journal: Cells

Article Title: Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines

doi: 10.3390/cells13161310

Figure Lengend Snippet: Colocalization of intracellular LAMP-1 with GrzB and the 15 kDa (RF10) or 9 kDa (pc) form of Gnly in NKL cells. Samples were processed as detailed in the legend of . ( A ) Histograms display the geometric mean value of the BDS score of respective stainings and the percentage of cells displaying a BDS score >2. ( B ) Representative images of stained cells. Scale bars represent 7 µm.

Article Snippet: For granulysin staining, we also used an unconjugated anti-Gnly mab (clone RF10, MBL International, Woburn, MA, USA) with Alexa-Fluor 555-conjugated goat anti-mouse polyclonal antibody (pab) (Thermo Fisher Scientific) and unconjugated anti-Gnly pab from goat (RD Systems, Minneapolis, MN, USA) with Alexa-Fluor 555-conjugated donkey anti-goat pab, respectively.

Techniques: Staining

Journal: Cells

Article Title: Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines

doi: 10.3390/cells13161310

Figure Lengend Snippet: NKL ( A , B ) or NK-92 ( C , D ) cells were stimulated or not for two hours with TPA and/or ionomycin with or without EGTA. The presence of 15 kDa and 9 kDa Gnly was assessed in cellular lysates and in precipitations with anti-Gnly (pc) from supernatants of unstimulated or stimulated cells. The blot was stained with the anti-Gnly (pc) to detect both forms of Gnly, as detailed in the Results section. ( A , C ) Representative Western blots. ( B , D ) Densitometric analysis of three independent experiments as depicted in ( A , C ) for 9 kDa Gnly and 15 kDa Gnly. Data are displayed as mean values ± standard deviation.

Article Snippet: For granulysin staining, we also used an unconjugated anti-Gnly mab (clone RF10, MBL International, Woburn, MA, USA) with Alexa-Fluor 555-conjugated goat anti-mouse polyclonal antibody (pab) (Thermo Fisher Scientific) and unconjugated anti-Gnly pab from goat (RD Systems, Minneapolis, MN, USA) with Alexa-Fluor 555-conjugated donkey anti-goat pab, respectively.

Techniques: Staining, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: TALEN-induced contraction of CTG trinucleotide repeats in myotonic dystrophy type 1 cells

doi: 10.1101/2023.10.14.562330

Figure Lengend Snippet: A: Design of the sgRNA and location of the PAM on the 3’ sequence of the DMPK gene. B: Transduction of GFP alone or Sa Cas9, its sgRNA and GFP in heterozygous transgenic mice fibroblasts carrying 250 CTG triplets. The CTG repeat was amplified with primers ST300f and ST300r. C: Quantifications of PCR signals from B. D: The Southern blot shows independent ASA clones in which the GFP alone or Sa Cas9, its sgRNA and GFP were transduced. Although small contractions or expansions could be detected in some clones, no significant differences were found between both experiments.

Article Snippet: For Cas9 lentiviral expression, we used a Sa Cas9 and GFP-containing backbone (Addgene #118836, plasmid pTRI211) into which was cloned the Sa Cas9-CTG sgRNA to give plasmid pTRI 212.

Techniques: Sequencing, Transduction, Transgenic Assay, Amplification, Southern Blot, Clone Assay