Journal: iScience

Article Title: Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells

doi: 10.1016/j.isci.2024.109119

Figure Lengend Snippet: Intracellular material acquisition from surrounding cells is specific to DCs and universal among all DC subsets tested (A) Splenic B cells (B), T cells (T), peritoneal macrophages (Mac), or dermal CD45 − cells (CD45 − ) were sorted from wild type C57BL/6 mice, labeled with SYTO62 and co-cultured with MutuDC1 cells for 45 min. Dots represent individual mice. Data combined from two experiments. (B) Sorted cells were labeled with CFSE and co-cultured for 45 min with SYTO62 labeled COCA keratinocytes (KC). Points represent individual mice. Data normalized and combined from two experiments. (C) Epidermal Langerhans cells (eLC), skin draining lymph node migratory Langerhans cells (sdLN LC), sdLN cDC1, sdLN cDC2, resident DCs (rDC), and mesenteric lymph node migratory DCs (mLN DC) were sorted from hLangCre-YFP f/f mice, labeled with CFSE, and co-cultured with RNA labeled COCA KCs for 45 min. Dots represent individual mice. Data pooled from three experiments. (D) moDCs were differentiated from human CD14 + monocytes and labeled with CFSE, then co-cultured with RNA labeled PBMCs on ice or 37°C for 45 min. Dots represent individual replicates. One representative experiment of two is shown. Data are represented as mean ± SD.

Article Snippet: SYTO62 Red Fluorescent Nucleic Acid Stain , Fisher Scientific , Cat# S11344.

Techniques: Labeling, Cell Culture

Journal: iScience

Article Title: Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells

doi: 10.1016/j.isci.2024.109119

Figure Lengend Snippet: Dendritic cells siphon RNA from neighboring cells through a contact dependent mechanism that does not resemble conventional means of antigen uptake (A) Outline of experiment to measure RNA and protein transfer to MutuDC1s with or without direct contact. (B) Flow cytometric analysis of SYTO62 RNA signal measured in MutuDC1s after 45 min incubation with RNA labeled COCA KCs. Results are from a single experiment. (C) Representative images and quantification of SYTO62 signal contained within MutuDC1s after keratinocyte:DC co-cultures. Mean pixel intensity of far-red channel (SYTO62) was calculated within the area occupied by GFP+ MutuDC1s on a per cell basis. Images acquired with a Nikon A1R confocal microscope using a Plan Fluor 40× Oil objective. Dots represent individual cells. Results are from a single experiment. (D) MutuDC1s (green) interacting with COCA KCs. Max projections of z stack images taken on a Nikon A1R confocal microscope using a Plan Fluor 40× Oil objective plus 10x scanner zoom. (E) Transfer of SYTO62 labeled RNA to MutuDC1s from keratinocytes relative to vehicle controls in the presence of an ATPsynthase inhibitor (1 μM Oligomycin A), an inhibitor of F-actin formation (8 μM Cytochalasin D), a macropinocytosis inhibitor (32 μM 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), and a gap junction inhibitor (5 mM 1-Heptanol). Data normalized and pooled from three experiments. Data are represented as mean ± SD.

Article Snippet: SYTO62 Red Fluorescent Nucleic Acid Stain , Fisher Scientific , Cat# S11344.

Techniques: Incubation, Labeling, Microscopy

Journal: iScience

Article Title: Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells

doi: 10.1016/j.isci.2024.109119

Figure Lengend Snippet: MutuDC1, but not MutuDC2 can cross-present the acquired ovalbumin (A) Control staining with SIINFEKL-MHC-I-specific antibody of B16, B16-OVA, MutuDC1, MutuDC1 pulsed with SIINFEKL, MutuDC2 and MutuDC2 pulsed with SIINFEKL. (B) MutuDC1 and MutuDC2 were co-cultured for the indicated time with B16 or B16-OVA and then the SIINFEKL-MHC-I levels determined by flow cytometry. Representative flow plots and summary graph (left lower corner) from one out of two experiments are shown with 2–3 technical replicates. (C) As in (B), but some of the MutuDC1 co-cultured with B16 or B16-OVA for 3 h were supplemented with PolyG/EDTA. Data from two independent experiments with 3 technical replicates were pooled. (D) MutuDC1 and MutuDC2 were co-cultured with B16 or B16-OVA labeled with SYTO62 for 45 min and then the transferred RNA signals (SYTO62) determined by flow cytometry. Data pooled from 3 independent experiments for B16, and one experiment for B16-OVA, with 2–3 technical replicates. Data are represented as mean ± SD.

Article Snippet: SYTO62 Red Fluorescent Nucleic Acid Stain , Fisher Scientific , Cat# S11344.

Techniques: Control, Staining, Cell Culture, Flow Cytometry, Labeling

Journal: iScience

Article Title: Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells

doi: 10.1016/j.isci.2024.109119

Figure Lengend Snippet: Inflammation does not alter RNA acquisition by DCs (A) MutuDC1s and MutuDC2s were exposed to 1 μg/mL LPS, 10 μg/mL IFNα, 5 μg/mL PolyI:C, or 0.5 μM CpG for 12 h and co-cultured with SYTO62-labeled B16 cells. The acquired RNA signal in DCs was determined by flow cytometry. (B) Like (A), but the B16 cells were treated as indicated. Data were pooled from two independent experiments, with 2–3 technical replicates. Data are represented as mean ± SD.

Article Snippet: SYTO62 Red Fluorescent Nucleic Acid Stain , Fisher Scientific , Cat# S11344.

Techniques: Cell Culture, Labeling, Flow Cytometry

Journal: iScience

Article Title: Dendritic cells overcome Cre/Lox induced gene deficiency by siphoning cytosolic material from surrounding cells

doi: 10.1016/j.isci.2024.109119

Figure Lengend Snippet:

Article Snippet: SYTO62 Red Fluorescent Nucleic Acid Stain , Fisher Scientific , Cat# S11344.

Techniques: Recombinant, Microscopy, Modification, Reverse Transcription, SYBR Green Assay, Saline, Staining, Isolation, Selection, Software, Electron Microscopy, Cell Culture, Sterility