Celltrace violet

Manufactured by Thermo Fisher Scientific

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CellTrace Violet is a cell tracking dye used for flow cytometry applications to monitor cell division. It binds to intracellular proteins, allowing the dye to be evenly distributed between daughter cells during cell division, enabling the visualization and quantification of cell proliferation.

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Lab products found in correlation

1 232 protocols using celltrace violet

High-Throughput Phenotyping of Engineered NK Cells

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For all experiments, flowcytometry was carried out using a MACSQuant Analyzer 10. Propidium iodide staining was employed to identify viable cells. Antibodies were from Miltenyi Biotec and included in addition to the human MACS marker screen monoclonal antibodies targeting CD3, CD19, CD56, and CX3CR1. CD19-CAR expression was detected by using a recombinant human CD19 Fc (R&D Systems) fusion protein at a concentration of 25 μg/mL followed by incubation with a monoclonal anti-Fc antibody conjugated to APC (Miltenyi Biotec). Of note, to remove nonspecific background staining from NK cells was minimized by incubation of the cells for 48 h in serum free culture medium prior to the staining. Where indicated, proliferation rates were determined by flow cytometry by labeling of the cells with CellTrace Violet (Thermo Fisher) at a concentration of 10 μM. High-throughput surface marker screening was carried out 3–4 days after transduction and CellTrace Violet labeled NK cells were incubated with the MACS Marker Screen human antibody panel targeting 371 surface markers. Detection was carried out by automated flowcytometry using a MACSQuant 10 Analyzer (Miltenyi Biotech).

Bari R., Granzin M., Tsang K.S., Roy A., Krueger W., Orentas R., Schneider D., Pfeifer R., Moeker N., Verhoeyen E., Dropulic B, & Leung W. (2019). A Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy. Frontiers in Immunology, 10, 2001.

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In vivo Cytotoxicity Assay for Antigen-Specific T Cells

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In vivo killing assay was performed as previously described (Kim et al., 2014 ). Briefly, CD45.1⁺ splenocytes were pulsed with 1 mg/ml of OVA_257–264 peptide or gp33 peptide or phosphate-buffered saline (PBS) at 37 °C for 1 h. These antigen- or PBS-pulsed splenocytes were then labelled with 0.5 μM of CellTrace Violet (CTV^lo) (Thermo Fisher Scientific) or 5 μM of CellTrace Violet (CTV^hi), respectively, at 37 °C for 15 min. The CTV^lo and CTV^hi labelled splenocytes were mixed at a 1:1 ratio and a total of 2×10⁷ cells were transferred to host mice that were challenged with Lm-OVA or LCMV at day > 35 prior, followed by analysis of in vivo cytotoxicity against these splenocytes after 2.5 h.

Huang H., Zhou P., Wei J., Long L., Shi H., Dhungana Y., Chapman N.M., Fu G., Saravia J., Raynor J.L., Liu S., Palacios G., Wang Y.D., Qian C., Yu J, & Chi H. (2021). In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions. Cell, 184(5), 1245-1261.e21.

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Antigen-specific CD8+ T cell activation

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Antigen-specific naive CD8⁺ T cells were isolated from the spleen of P14 TCR-transgenic mice and purified by negative selection using magnetic-activated cell sorting (Miltenyi Biotec). T cells were then labeled with 5 μM CellTrace™ Violet (Thermo Fisher) and cocultured for 3 days with either BMDCs or SPDCs at a ratio of 5:1 (500,000 T cells:100,000 DCs). For stimulation, 20 ng/ml GP_33-41 peptide and GolgiPlug/GolgiStop™ (BD Bioscience) were added to the cultured cells for the last 5 hours. CD8⁺ OT-I T cells were isolated from the spleen of OT-I transgenic mice and purified by negative selection with MagniSort™ beads (Thermo Fisher). The T cells were then labeled with 5 μM CellTrace™ Violet (Thermo Fisher) and cocultured for 3 days with either BMDCs or SPDCs pulsed for 2 hours with the OVA protein (20 μg/ml) at a ratio of 5:1 (250,000 T cells:50,000 DCs). For stimulation, 0.5 μg/ml OVA_257-264 SIINFEKL peptide and GolgiPlug/GolgiStop™ (BD Bioscience) were added to the cultured cells for the last 5 hours.

Yoo S., Jeong Y.H., Choi H.H., Chae S., Hwang D., Shin S.J, & Ha S.J. (2023). Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells. Experimental & Molecular Medicine, 55(5), 999-1012.

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Antibody-Dependent Phagocytosis Assay

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Porcine monocytes were enriched from fresh PBMCs by removing CD3ϵ positive cells using a MACS cell separation LD column (Miltenyi Biotec). The monocyte containing fractions were incubated with 50 ng/mL of human M-CSF (Miltenyi Biotec) for 6 days to differentiate them into macrophages. MDCK-HA target cells were fluorescently labelled with CFSE (ThermoFisher) following the manufacturer’s instructions and incubated with serially diluted pb27/pb39 IgG subclasses or anti-Nipah virus G protein IgG1 mAb as negative control for 30 min at RT. Differentiated macrophages were fluorescently labelled with CellTrace Violet (ThermoFisher) and added at an effector to target ratio of 20:1, in AIM-V medium at 2 × 10⁶ cells/mL. ADCP was determined after 3 h of culture by flow cytometry as described above measuring the percentage of live macrophages that contained target cells indicated as double positive for CellTrace Violet and CFSE.

Paudyal B., Mwangi W., Rijal P., Schwartz J.C., Noble A., Shaw A., Sealy J.E., Bonnet-Di Placido M., Graham S.P., Townsend A., Hammond J.A, & Tchilian E. (2022). Fc-Mediated Functions of Porcine IgG Subclasses. Frontiers in Immunology, 13, 903755.

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Treg Suppression Assay Protocol

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The suppressor activity of expanded Tregs was analyzed using co-culture assays. Human PBMCs (Lonza) were labeled with Cell Trace Violet (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Expanded Tregs were cultured with Cell Trace Violet-labeled PBMCs at different ratios together with Dynabeads human T-activator CD3/CD28 (Thermo Fisher) for 3 days. Proliferated PBMCs were stained with anti-CD4-PEcy7 (BioLegend, San Diego, CA, USA). The proliferation of CD4⁺ T cells was analyzed using a BD FACSAria (BD Biosciences, San Jose, CA, USA). The sigmoid curves of Treg suppression functions were visualized in R using the ggplot2 package (27 ) and the nls2 package (29 ).

Okuzono Y., Miyakawa S., Itou T., Sagara M., Iwata M., Ishizuchi K., Sekiguchi K., Motegi H., Oyama M., Warude D., Kikukawa Y, & Suzuki S. (2024). B-cell immune dysregulation with low soluble CD22 levels in refractory seronegative myasthenia gravis. Frontiers in Immunology, 15, 1382320.

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CD4 T-cell Proliferation Assay

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To assess the effect of IL-10 on CD4 T-cell proliferation, cell trace violet (Thermofisher Scientific) was used as per manufacturer recommendation. CD4 + T cells isolated from PBMCs were stained with 5 µM cell trace violet (Thermofisher Scientific), following the manufacturer’s instructions. Cells were then cultured with varying concentrations of CD19 + B cells from 0.6 × 10⁵–0.24 × 10⁶/well. They were subsequently cultured in 96-well flat-bottom plates, containing plate-bound CD3 and CD28 (both 1 mg/ml), at 0.3 × 10⁶ cells per well. After 4 days, cells were harvested and acquired for staining and flow cytometry.

Patel A.J., Willsmore Z.N., Khan N., Richter A., Naidu B., Drayson M.T., Papa S., Cope A., Karagiannis S.N., Perucha E, & Middleton G.W. (2022). Regulatory B cell repertoire defects predispose lung cancer patients to immune-related toxicity following checkpoint blockade. Nature Communications, 13, 3148.

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Single-cell metabolic analysis protocol

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Cells were stained with either CellTrace™ Violet (Thermo Fisher, C34571), CellTrace™ Far Red (Thermo Fisher, C34564), or pHrodo™ Green AM (Thermo Fisher, P35373) at 2 × 10⁶ cells per mL. CellTrace™ Violet (5 μM) and pHrodo™ Green AM (10 μM, addition of 1X PowerLoad™) stainings were performed in HBSS for 30 min on ice, CellTrace™ Far Red (1 μM) staining in HBSS for 10 min on ice. After incubation, the cells were washed with ice-cold MACS buffer. The cells were collected (400 g, 5 min, 4 °C) and re-suspended in ice-cold assay buffer (RPMI 1640 without phenol red, #11835063, 10% (v/v) KnockOut Serum Replacement, #10828010, 1X penicillin–streptomycin, #10378016, 25 mM MOPS pH 7.5, #J61843, 0.1% (v/v) Pluronic F-127, #11835030, all Thermo Fisher, and 0.5% (w/v) recombinant human serum albumin, Sigma Aldrich, A9731) to achieve a λ (mean number of cells per droplet) of 0.25–0.50.
Lactate standards (Lactate Assay Kit, Sigma-Aldrich, MAK064) with concentrations from 2 to 200,000 amol/nL were prepared. IgG (Kerafast, EFD006) and IgM isotype controls (Biolegend, 401,601) of 100 nM were prepared, with eight serial dilutions by a factor 2.

Bucheli O.T., Rodrigues D., Portmann K., Linder A., Thoma M., Halin C, & Eyer K. (2024). Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis. Scientific Reports, 14, 8507.

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Phagocytosis Assay of Macrophages

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Macrophages generated as above were harvested from plates using Trypsin-EDTA (Thermo fisher scientific). The indicated target cells were labeled with Celltrace violet (Thermo fisher scientific) according to the manufacturer’s protocol, and phagocytic assay was performed by co-culturing 6×10⁴ Celltrace violet-labeled target cells with 3×10⁴ human macrophages for 2 hr in ultra-low attachment 96-well flat bottom plates in IMDM + GlutaMax without antibiotics or serum added. All cells were harvested after coculture, and phagocytosis was determined by flow cytometry analyses, in which the phagocytic ratio is calculated as the percentage of macrophages that engulfed target cells (human CD45⁺CD14⁺Celltrace violet⁺) among total macrophages (human CD45⁺CD14⁺).

Li Y., Wu Y., Federzoni E.A., Wang X., Dharmawan A., Hu X., Wang H., Hawley R.J., Stevens S., Sykes M, & Yang Y.G. (2022). CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals. eLife, 11, e73677.

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Naive CD44lo LN T Cell Division

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Naïve CD44^lo LN T cells were labeled with Cell Trace Violet (Thermo Fisher) and incubated with recombinant IL-2 (0.5 μg/ml), IL-7 (0.5 μg/ml) or IL-15 (0.5 μg/ml) for 5 days. Cell division was assessed by flow cytometry for Cell Trace Violet dye dilution per the manufacturer’s recommended protocol (Thermo Fisher).

Keller H.R., Kim H.K., Jo Y., Gress R.E., Hong C, & Park J.H. (2020). The abundance and availability of cytokine receptor IL-2Rβ (CD122) constrain the lymphopenia-induced homeostatic proliferation of naïve CD4 T cells. Journal of immunology (Baltimore, Md. : 1950), 204(12), 3227-3235.

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Splenic Cell Proliferation and Migration

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Splenic cells were cultured in lymphocyte media (RPMI with 55 μM 2-mercaptoethanol and 10% fetal calf serum), and incubations were at 37°C with 5% CO₂. For testing T cell proliferation, total splenic cells were labeled with CellTrace Violet (Thermo Fisher) and stimulated with 1 μg/mL plate-bound anti-CD3 and 0.25 μg/mL soluble CD28 (clones OKT-3 and 9.3, respectively; University of California, San Francisco Monoclonal Antibody Core), followed by a 72-hr incubation and then flow cytometry for WASp expression and CellTrace Violet dilution. As a positive control, some cells were stimulated with 2 ng/mL PMA and 500 ng/mL ionomycin. The B cell migration assays were performed using CD43-depleted splenocytes (CD43 microbeads; Miltenyi Biotec). One-hundred microliters of cells in lymphocyte media (1 × 10⁶/mL) was seeded in duplicate upper wells of a 5-μm transwell plate (Corning Costar). The lower wells had 600 μL lymphocyte media and 300 nM recombinant murine CXCL13 (R&D Systems). Following a 1-hr incubation, the numbers of cells migrating to the lower well were counted, and the percentage of cells migrating was calculated based on the input cell number.

Singh S., Khan I., Khim S., Seymour B., Sommer K., Wielgosz M., Norgaard Z., Kiem H.P., Adair J., Liggitt D., Nienhuis A, & Rawlings D.J. (2016). Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector. Molecular Therapy. Methods & Clinical Development, 4, 1-16.

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