Journal: Journal of Cellular and Molecular Medicine

Article Title: Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR

doi: 10.1111/j.1582-4934.2009.00784.x

Figure Lengend Snippet: Workflow of the procedure. Briefly, cytospin preparations on membrane-coated slides are fixed and stained using immunocytochemistry and DNA counterstaining. Automated scanning and eventual relocation of positive candidate cells facilitate their microdissection and laser catapulting onto water droplets on anchors of AmpliGrid slides. After evaporation of the water, the cells are lysed and multiplex PCR is performed in droplets on the slide anchors. Finally, the amplification products are forwarded to analysis by capillary electrophoresis.

Article Snippet: The anchors were checked for the presence or absence of microdissected cells and forwarded to cell lysis, followed by single and pooled cell DNA fingerprinting using a commercial multiplex PCR kit (PowerPlex 16 System Kit; Promega, Mannheim, Germany) that co-amplifies 15 polymorphic STR loci and the amelogenin locus.

Techniques: Membrane, Staining, Immunocytochemistry, Laser Capture Microdissection, Evaporation, Multiplex Assay, Amplification, Electrophoresis

Journal: Journal of Cellular and Molecular Medicine

Article Title: Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR

doi: 10.1111/j.1582-4934.2009.00784.x

Figure Lengend Snippet: DNA profiles amplified from cells microdissected from sample 3. Top: single GZ 158-positive JAR cell (as shown in , top right). Bottom: cell pool of PBMNC to which the anti-trophoblast antibody GZ 158 did not bind. PCR products allowing unambiguous allocation of cells are highlighted with red (PBMNC) or green (JAR cell) triangles. Loci that show uninformative PCR products matching both DNA profiles are indicated with red-green striped triangles. Black triangles indicate allele drop-out at the respective loci as compared with DNA profiles derived from the summary of individual DNA fingerprinting from the respective individuals/samples.

Article Snippet: The anchors were checked for the presence or absence of microdissected cells and forwarded to cell lysis, followed by single and pooled cell DNA fingerprinting using a commercial multiplex PCR kit (PowerPlex 16 System Kit; Promega, Mannheim, Germany) that co-amplifies 15 polymorphic STR loci and the amelogenin locus.

Techniques: Amplification, Derivative Assay, DNA Profiling

Journal: Genome Medicine

Article Title: Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

doi: 10.1186/s13073-020-00756-z

Figure Lengend Snippet: Combined single-cell transcriptional and proteomics immunophenotyping provides a high-resolution map of human primary CD4 + T cells in the blood. a Summary of the experimental workflow. FACS plot depicting the sorting strategy for the isolation of the three assessed CD4 + T cell populations. b Two-dimensional plot depicting the expression of IL-7R and IL-2RA at the protein level using oligo-conjugated antibodies (AbSeq). Cells are coloured according to their respective sorting gate, as assessed using oligo-conjugated sample-tagging antibodies. c Uniform Manifold Approximation Projection (UMAP) plot depicting the clustering of all captured CD4 + single cells using the combined proteomics and transcriptomics data. Expression levels of the CD45RA (black to green) and CD45RO (black to red) isoforms obtained using the AbSeq technology are depicted in the plot. d UMAP plot depicting the clustering of resting primary CD4 + T cells ( n = 9708) isolated from the blood of a systemic lupus erythematosus (SLE) patient. Dashed lines delineate the naive Teff (black), memory Teff (red), and Treg (blue) clusters, annotated manually based on their respective protein and mRNA expression profiles. e Heatmap displaying the top 10 differentially expressed genes in each resting CD4 + Teff cluster. f UMAP plots depicting the expression of the CD4 + T cell lineage-defining transcription factors TBET (Th1) and RORγt (Th17) in resting CD4 + T cells. Arrows recapitulate the identified axis of Th1 and Th17 differentiation and are supported both on the gradient of expression of the respective lineage-restricted transcription factors (TBET and RORγt, respectively) and on the developmental trajectories identified by the pseudotime analysis depicted in Fig. . g Expression of the effector-type cytokine transcripts IFNG , NKG7 , PRF1 , CCL5 , GZMH and GZMK in resting CD4 + T cells

Article Snippet: Barcoded oligo-conjugated antibodies (single-cell multiplexing kit; BD Biosciences) were used to infer the origin of sample (i.e. sorted cell population) and multiplet rate by the BD Rhapsody Analysis pipeline.

Techniques: Isolation, Expressing