celltrace violet  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Potential of HLA-E-targeting diabodies to induce lysis of HIV-1-infected cells by CD8 + T cells

doi: 10.64898/2026.04.28.721204

Figure Lengend Snippet: Specificity of RLP-13 in dual-color co-cultures. (A) Setup of dual-color co-cultures: K562 cells transfected with covalently linked peptide:HLA-E expression constructs, presenting either cognate (Mtb44) or non-cognate (SP-2A) peptide, are stained with CellTrace Violet (CTV) or CFSE, respectively, and cultured for 18 hours with pre-expanded CD8 + T cells and different concentrations of RLP-13 at a ratio of 4:1:3 (Effector : Cognate target : Non-cognate target). Cells are then stained with lineage and activation markers and viability dye for analysis by flow cytometry. (B) Representative flow plot of viable single cell populations from co-culture wells without (left) or with (right) RLP-13. Indicated cognate and non-cognate gated population frequencies were used to calculate the relative viability of target cell populations. (C) Target cell viability of indicated cognate and non-cognate populations from co-cultures with various concentrations of either RLP-13 (red and pink lines) or irrelevant control scDb H2-mu (black and green lines). Viability was calculated by normalizing the frequency of the indicated target cell populations to that observed in co-culture wells without any scDb added. Assay was repeated five independent times across two HIV-negative donors. Shown measurements are averaged over eight technical replicates from one representative experiment. (D) Concentrations of the indicated effector molecules in the supernatants of co-cultures shown in (C) measured using the LegendPlex CD8/NK cell cytokine panel kit.

Article Snippet: For dual-color co-cultures, target cells were stained with CellTrace Violet or CFSE (dilution 1:20,000, FisherScientific, Cat. # C34557 and Cat # C34554) for 15 minutes in PBS at 37°C prior to plating.

Techniques: Transfection, Expressing, Construct, Staining, Cell Culture, Activation Assay, Flow Cytometry, Single Cell, Co-Culture Assay, Control

Journal: ImmunoHorizons

Article Title: Maturation of thymocytes with a monoclonal TCR under control of Trac promoter elements in the absence of β-selection

doi: 10.1093/immhor/vlaf035

Figure Lengend Snippet: 1045 TRex Tcrb −/− CD8 + T cells populate the periphery and are functional. (A) Spleen weights from mice with indicated 1045 and Tcrb genotypes. (B, C) CD8 + T cell frequency of CD45 + splenocytes (B) or number per mg of spleen (C). (D, E) Representative plots (D) of Vβ9 + frequency of CD8 + T cells (E). (F) Vβ9 gMFI on CD8 + T cells. G) CD44 + frequency of Vβ9 + CD8 + T cells. (H–J) Representative plots (H) of γδTCR + CD4 − CD8 − frequency of CD45 + splenocytes (I) or number per mg of spleen (J). (K, L) Representative plots (K) of Vβ9 + and γδTCR + frequency of CD8 + or CD4 + T cells (L). (M) Number of Vβ9 + CD8 T cells from 1045 +/+ Tcrb + or 1045 +/+ Tcrb −/− splenocytes following 72 h of stimulation with varying concentrations of Msln 406–414 peptide. (N) Representative histograms of proliferation of Vβ9 + CD8 + T cells stimulated with 0.1 µg/mL Msln 406–414 peptide as assessed by CellTrace Violet (CTV) dilution. (O) Frequency of Vβ9 + CD8 + T cells by number of divisions in splenocytes stimulated with 0.1 µg/mL Msln 406–414 peptide. (P) Frequency of Vβ9 + CD8 + T cells that reached ≥4 divisions across varying concentrations of Msln 406–414 peptide. (Q) Schematic depicting in vitro priming and restimulation of 1045 +/+ Tcrb + or 1045 +/+ Tcrb −/− splenocytes for evaluation of cytokine production. (R) Representative plots of IFNγ, TNFα, and granzyme B (GzmB) production in Vβ9 + CD8 + T cells following restimulation with 0.001 µg/mL Msln 406–414 peptide. (S–U) IFNγ + (S), IFNγ + TNFα + (T), and GzmB + (U) frequency of Vβ9 + CD8 + T cells following restimulation with varying concentrations of Msln 406–414 peptide. (A–L) Each dot represents an individual animal with n = 3 to 8 animals per group and mean ± SEM shown. (M–U) Mean ± SD of technical triplicates, representative of 3 independent experiments. Comparisons evaluated using 1-way analysis of variance with Tukey posttest in panels A to J and unpaired Student’s t test in panels L to U. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then stained with 1 μM CellTrace Violet (Thermo Fisher Scientific) in PBS for 20 min at 37 °C and subsequently quenched with 4 volumes of 4 °C T cell media.

Techniques: Functional Assay, In Vitro