celltrace tm violet cell proliferation kit  (Thermo Fisher)

Journal: Cell reports

Article Title: Transplants foster B cell alloimmunity by relaying extracellular vesicles to follicular dendritic cells

doi: 10.1016/j.celrep.2025.115832

Figure Lengend Snippet: (A) Donor H2 Ag in splenic FDCs (inset, arrows) after transplantation of B6 (H2 b ) hearts in BALB/c mice. Original magnification ×200. Scale bars, 20 μm. (B) Quantification with ImageJ of donor H2 Ag spots per FDC network on spleens of BALB/c mice transplanted with B6 hearts. (C) Donor H2 Ag spots per FDC network on BALB/c LNs draining B6 skin allografts assessed using ImageJ. (D) Deep SIM of donor H2 Ag spots in FDCs of a BALB/c LN draining a B6 skin graft. Scale bar, 2 μm. ROI, region of interest. (E and F) 2P-microscopy of FDCs in the spleen of a BALB/c mouse transplanted with a B6 heart releasing RFP-sEVs (E) and a BALB/c LN draining a B6 skin graft releasing RFP-sEVs (F). Arrows: graft-RFP-sEVs on the surface (red) or inside (yellow) of FDCs (green). CellTrace Violet-B cells indicate B cell follicles. FDCs were labeled with AF488-CD21/CD35 Ab. Original magnification ×25. Scale bars, 20 μm. (G) Colocalization by confocal microscopy of donor (B6, IA b + H2K b ) allo-Ags in FDCs of LNs draining CMV Cre/+ LSL -RFP-CD63 B6 skin grafted in BALB/c mice. Scale bar, 10 μm. Mander’s colocalization coefficient of graft-sEVs and donor IA b + H2K b Ab analyzed using Imaris. Each triangle represents one FDC network. In (E) and (F), RFP spots per FDC network z stacks were quantified within 90-μm-thick z stacks using Imaris. Experiments were done on 4 spleens per POD (A and B), 6 graft-dLNs per POD (C), 3 graft-dLNs (D), 4 spleens (E), 4 graft-dLNs (F), and 12 graft-dLNs (G). In (B), (C), (E), and (F), comparisons were made by multiple unpaired two-tailed Student’s test. Error bars, means ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; NS, not significant.

Article Snippet: CellTrace TM Violet Cell Proliferation Kit, for flow cytometry , Thermo Fisher , Cat # C34557.

Techniques: Transplantation Assay, Microscopy, Labeling, Confocal Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: LAG3 marks activated but hyporesponsive NK cells

doi: 10.1101/2024.12.18.629184

Figure Lengend Snippet: (A) C57BL/6 mice (n=4 for each day) were injected intraperitoneally with 200ug of Poly (I:C) and sacrificed in kinetic. The percentage of Sca1+, LAG3+ and CD69+ splenic NK cells was analyzed by flow cytometry. (B) The percentage of LAG3+ among Sca1+ and Sca1-NK cells on Day2 was analyzed by flow cytometry (n=5). (C-G) C57BL/6 mice (n=2 for each experiment) were injected intraperitoneally with 200ug of Poly (I:C) and sacrificed on Day2. Splenic NK cells were stained for the phospho-epitope pS6 Ser235/236 (C) and for pAkt S473 (D). Overlays of representative histograms are shown (left panels). The MFI values are indicated on the graph and represented on the right panel for each individual, n=6, 3 experiments in total. (E) NK cells were stained with Mitotracker Green and CellRox. The MFI values for each mouse (n=7, 3 experiments in total) for the analyzed marker are represented. (F) The MFI of CD98 (left) or CD71 (right) was determined by flow cytometry on LAG3+ and LAG3-NK cells (n=3). Due to the small sample size, we did not calculate p-values. (G) SSC-A and FSC-A parameters on NK cells were measured by flow cytometry in LAG3- and LAG3+ NK cells (n=5). (H) Splenic NK cells from C57BL/6 mice (n=6, 3 experiments) were isolated, stained with Cell Trace Violet (CTV) and cultured with 100ng/mL of IL-15 for 3 days. The MFI of CTV was analyzed by flow cytometry and the overlays of representative histograms are shown (left panel). The percentage of divided NK cells for each individual was calculated using the proliferation modeling tool on the FlowJo software. All the data were analyzed using a paired non-parametric Wilcoxon test. p-values are indicated on each graph.

Article Snippet: Isolated NK cells were stained with 0.6µM Invitrogen CellTrace TM Violet (CTV) Cell Proliferation Kit according to manufacturer’s instructions.

Techniques: Injection, Flow Cytometry, Staining, Marker, Isolation, Cell Culture, Software