Journal: Cell reports

Article Title: Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

doi: 10.1016/j.celrep.2021.109105

Figure Lengend Snippet: (A) A generalized schematic of the platform. Human CD14 + monocytes are isolated from blood by density gradient separation of PBMCs followed by magnetic negative selection. Either PBMCs or monocytes may be cryopreserved for later editing . Cells are then nucleofected with preformed CRISPR-Cas9 RNPs and immediately put into differentiating culture under MDM- or MDDC-generating conditions. After allowing for 6–7 days of differentiation and washout of the targeted gene product, cells can be subjected to a wide variety of functional, phenotypic, and genotypic studies to assess the knockout efficiency and function of the targeted gene product. (B) Guide sequence-dependent knockout of targeted genes leads to loss of gene products. CD14 + monocytes were nucleofected with RNPs containing 1 of 5 distinct guide sequences against the indicated gene or a scrambled non-targeting control, cultured under MDM-generating conditions, and then lysed for immunoblot analysis. Blots show targeted gene protein product and untargeted housekeeping gene product β-actin protein levels in cells from 2 blood donors. GNE1 and ATP6V1A ran at their expected sizes of 79 and 69 kDa, respectively. (C) Knockout was quantified by digital densitometry and normalized on a per-sample basis in relative fluorescence units (RFUs) to untargeted housekeeping control protein β-actin. See also .

Article Snippet: To assess the quality of the CD14+ negative selection, samples before and after CD14 enrichment were stained with antibodies against CD14-PE (1:25)(Miltenyi 130–110-519) and CD16-APC (1:25)(Miltenyi 130–106-705).

Techniques: Isolation, Selection, CRISPR, Functional Assay, Knock-Out, Sequencing, Control, Cell Culture, Western Blot, Fluorescence

Journal: Cell reports

Article Title: Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

doi: 10.1016/j.celrep.2021.109105

Figure Lengend Snippet: (A) Principal-component analysis of RNA sequencing (RNA-seq) from the indicated cell types. (B) Normalized transcript abundance ( Z score) for selected markers of MDM or MDDC differentiation . (C) Dendrogram of hierarchical clustering of the data in (B) by Euclidean distance. (D and E) Among cells subjected to CRISPR-Cas9 RNP nucleofection, cell surface protein levels of CD16, CD14, and CD206 were compared between the cells that bear the desired β2 m knockout (pink) and those that do not (teal) by flow cytometry after 7 days of MDM differentiation. (D) shows gating, while (E) shows the expression of the indicated markers. (F) Representative images of unperturbed (left) and RNP-nucleofected (right) MDMs infected with GFP-expressing M. tuberculosis (Mtb-GFP) show that CRISPR-Cas9-targeted cells remain competent to phagocytose living pathogens. Top, membrane staining with Cell Mask Far Red; CENTER, Mtb-GFP; bottom, composite. Scale bars represent 100 μm. See also .

Article Snippet: To assess the quality of the CD14+ negative selection, samples before and after CD14 enrichment were stained with antibodies against CD14-PE (1:25)(Miltenyi 130–110-519) and CD16-APC (1:25)(Miltenyi 130–106-705).

Techniques: RNA Sequencing, CRISPR, Knock-Out, Flow Cytometry, Expressing, Infection, Membrane, Staining

Journal: Cell reports

Article Title: Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins

doi: 10.1016/j.celrep.2021.109105

Figure Lengend Snippet:

Article Snippet: To assess the quality of the CD14+ negative selection, samples before and after CD14 enrichment were stained with antibodies against CD14-PE (1:25)(Miltenyi 130–110-519) and CD16-APC (1:25)(Miltenyi 130–106-705).

Techniques: Blocking Assay, Recombinant, Virus, Purification, Staining, Isolation, Cell Viability Assay, Plasmid Preparation, Software, Cell Analysis, Modification, Membrane, Western Blot, Cell Culture, Sterility

Journal: bioRxiv

Article Title: Interaction between TREM2-Macrophages and Cutibacterium acnes Drives Altered Lipid Metabolism in Chronic Apical Periodontitis

doi: 10.64898/2026.04.19.719526

Figure Lengend Snippet: a. Detection of CA in CAP lesions. Representative periapical radiograph showing a radiolucent lesion (white dotted line) at the apex of tooth #22 (left panel). Representative IHC staining using a PAB antibody revealed the presence of CA as small punctate signals within the cytoplasm of inflammatory cells (red dotted circles). (Scale bar, 30 μm) b. Multiplex immunohistochemical (mIHC) analysis of CA localization in M □. mIHC was performed using antibodies against CD14 (surface marker), CD68 (endolysosomal marker), and CA (PAB) in retrospective FFPE tissues from seven cases of CAP. Representative images show CA signals (red) localized within CD14□ and/or CD68□ cells (white dotted circles), as well as signals detected outside CD14□/CD68□ cells (yellow dotted circle). The proportion of CA in M□ (CD14+/CD68+, CD14-/CD68+, CD14+/CD68-) and out of M□ (CD14-/CD68-) was automatically counted as described in Methods (right panel). Note a few proportion of CA is localized in outside of M□ (black). (Scale bars, 30 μm) c. Validation of intracellular CA in M □ using a strain-specific antibody. Schematic illustration of the experimental workflow for enrichment of CD14□ monocyte/macrophage populations from CAP tissues, followed by immunofluorescence (IF) staining (upper panel). CAP samples were dissociated into single cells, and CD14□ cells were isolated by MACS prior to IF analysis. Representative confocal images show CA signals detected using both PAB and strain-derived polyclonal anti-CA antibodies within CD68□ cells. (Scale bars, 20 μm)

Article Snippet: Monocyte-lineage cells were enriched using CD14 MicroBeads (Miltenyi Biotec, #130-097-052) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Multiplex Assay, Immunohistochemical staining, Marker, Biomarker Discovery, Immunofluorescence, Staining, Isolation, Derivative Assay