Journal: Nature neuroscience

Article Title: Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB.

doi: 10.1038/s41593-021-00864-y

Figure Lengend Snippet: Fig. 3 | Transition of GFAP:tdTomato+ cells into new neurons results in no depletion in the total GFAP cell number. a, Images of newly induced neurons (iNeurons) expressing NeuN (green) and tdTomato (red) (NeuN visualized by IF and tdTomato by direct fluorescence); blue, DAPI staining for DNA; white, GFAP visualized by IF; the experiment was reproduced four times, independently, with similar results. b, Total number of tdTomato+NeuN− cells (left) or tdTomato+NeuN+ cells (right), either with or without GFAP labeling, per dentate gyrus, 2 months after ICV injection of PTB-ASO. Data are presented as mean ± s.e.m. (left: tdTomato/NeuN: 176.8 ± 34.92; tdTomato/NeuN/GFAP mean: 167 ± 34.32; right: tdTomato/NeuN mean: 28.88 ± 4.87; tdTomato/NeuN/GFAP mean: 0.87 ± 0.12; n = 4 biological repeats; two-tailed t-test **P = 0.0012). c, Representative images of human organoids 1 month after PTB-ASO or control-ASO application in the growth media GFAP (green) and Nestin (red) (both visualized by IF); blue, DAPI staining for DNA; the experiment was reproduced three times, independently, with similar results. d, Representative images of a dentate gyrus taken 2 months after ICV injection of control or PTB-ASO2 into 3-month-old and 1-year-old mice; white, GFAP visualized by IF; the experiment was reproduced three times for control and aged mice and four times for 5-month-old mice, independently, with similar results. e, Total GFAP-positive cells per dentate gyrus area of 5-month- and 1.2-year-old PTB-ASO- or control-injected mice. Data are presented as mean ± s.e.m. (control mean: 1,074 ± 83.55; 5 months PTB-ASO mean: 1,006 ± 82.45; 1.2 year-old PTB-ASO mean: 1,214 ± 104.8; n = 3, n = 4, n = 3 biological repeats).

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Rabbit-PTBP1 (Abclonal; Cat# - A6107; IF concentration - 1:1000); Mouse-GFAP (Dako; Cat# - M0761; IF concentration - 1:500); Mouse-NeuN (Millipore; Cat# - MAB377; IF concentration - 1:100); Rabbit-pan-ASO (Ionis pharmaceutical, homemade antibody; Cat# -13545; IF concentration - 1:200); Chicken-MAP2 (Abcam; Cat# - ab5392; IF concentration - 1:1000); Mouse-Nestin (abclonal; Cat# - AB22035; IF concentration - 1:200); Rabbit-GFAP (Novus Biologicals; Cat# - NB300-141; IF concentration - 1:1000); Mouse-TubIII (Tuj1) (Millipore Sigma; Cat# - T8660: IF concentration - 1:500); Goat-DCX (Santa Cruz Biotechnology; Cat# - Sc-8066; IF concentration - 1:500).

Techniques: Expressing, Fluorescence, Staining, Labeling, Injection, Two Tailed Test, Control

Journal: Scientific Reports

Article Title: Combined microfluidic enrichment and staining workflow for single-cell analysis of circulating tumor cells in metastatic prostate cancer patients

doi: 10.1038/s41598-024-68336-4

Figure Lengend Snippet: In-cassette staining method for CTC detection. ( A ) Schematic overview of the workflow. Abbreviations: mononuclear cells (MNC), copy-number alterations (CNA) ( B ) Recovery of 100 PC3 cells spiked into 10 mL healthy donor blood. The samples were processed with DPBS or DPBS + 1% BSA + 1 mM EDTA buffer on the Parsortix. CK Pos , DAPI Pos and CD45 Neg cells were manually counted as CTCs, and the recovery was calculated from the number of spiked cells vs. number of detected cells. Data is presented as mean ± sd. The comparison between the two groups was done using a Mann–Whitney U test ( p = 0.019) ( C ) Test of two different priming and separation buffers: DPBS or DPBS + 1% BSA + 1 mM EDTA. The harvested cells were plated on microscopy slides and the total number of DAPI Pos cells were automatically counted. The harvested cell fraction was calculated from the total number of cells (harvested fraction + cells left in the cassette). Data is presented as mean ± sd. Statistical analysis was performed using Mann–Whitney U test ( p = 0.017) ( D ) Spike in of 100 prostate cancer cells (PC3, LnCAP, DU145, C4-2) as described in B ( E ) Recovery of 10 PC3 cells spiked into 10 mL healthy donor blood ( F ) Stability of 100 PC3 cells spiked into 10 mL of healthy blood (CellRescue). The blood was processed after ~ 1 h, 24 h ( p = 0.062) or 72 h ( p = 0.024) and stained as described in B. The comparison between the three groups was performed using a Mann–Whitney U test ( G ) Recovery of 100 PC3 cells spiked into a K 2 EDTA blood tube and processed after 1 h. Data is presented as mean ± sd. ( H ) Staining of four prostate cancer cell lines (PC3, LNCaP, DU145 and C4-2) with DAPI and anti-CK. The cells were images by imaging flow cytometry (ImageStream).

Article Snippet: The captured cells in the cassette were incubated with permeabilization buffer (20 min) containing separation buffer + 0.15% saponin (Sigma-Aldrich, 47036) followed by immunostaining (90 min) with antibodies against cytokeratin (FITC-conjugated, Macs Miltenyi biotech, 130-118-964, clone CK36H5, 1:100), CD45 (Alexa Fluor 647-conjugated, eBioscience, 51-0459-42, clone HI30, 1:100) and DAPI (Fischer Scientific, D1304, 1:10.000) diluted in separation buffer containing 0.05% saponin.

Techniques: Staining, Comparison, MANN-WHITNEY, Microscopy, Imaging, Flow Cytometry