Celltrace violet cell proliferation kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, United Kingdom, Germany

The CellTrace Violet Cell Proliferation Kit is a laboratory instrument used to effectively track and measure cell division in various cell types. It provides a reliable and quantitative method for analyzing cell proliferation dynamics.

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CellTrace Violet (1232 citations) CellTrace Violet Proliferation Kit (42 citations) CellTrace™ Cell Proliferation Kit (35 citations) CellTrace™ Violet (CTV) Cell Proliferation Kit (26 citations) Cell Proliferation Kit (25 citations) CellTrace Proliferation Kit (10 citations) CellTrace Violet kit (3 citations) CellTrace CFSE or Violet (CTV) Cell Proliferation Kit (2 citations)

267 protocols using celltrace violet cell proliferation kit

NK Cell-Mediated Killing of CLL Cells

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CLL cells were stained with Cell Trace™ Violet Cell Proliferation Kit (Invitrogen™) then treated for 24-hours with selinexor (Karyopharm Therapeutics), leptomycin B (Sigma), ibrutinib (Selleckchem) or acalabrutinib (Selleckchem). CLL cells were treated with the caspase inhibitor Q-VD-OPh (QVD, 30 µM, Sigma) for 30 min prior to addition of anti-cancer agents. CLL cells were then co-cultured with healthy NK cells at an effector:target (E:T) ratio of either 1:1 or 5:1 for 4 h at 37 °C. After co-culture, cells were stained with propidium iodide (Invitrogen™) and NK cell-specific lysis of CLL cells assessed on a BD FACS Aria II (BD Biosciences) using FACSDiva software (BD Biosciences) and analysed with FlowJo v10.7.1 (BD Biosciences). NK cell-specific Lysis was calculated as follows:

N K s p e c i f i c l y s i s = \frac{(%CLL lysis in coculture) - (%spontaneous CLL lysis)}{Maximum lysis - % spontaneous CLL lysis} * 100

Fisher J.G., Doyle A.D., Graham L.V., Sonar S., Sale B., Henderson I., Del Rio L., Johnson P.W., Landesman Y., Cragg M.S., Forconi F., Walker C.J., Khakoo S.I, & Blunt M.D. (2023). XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression. Leukemia, 37(10), 2036-2049.

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Immune Cell Proliferation and Cytokine Assays

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T cell proliferation was quantified using dilution of CellTrace™ Violet Cell Proliferation Kit (Invitrogen, Waltham, MA, USA) after stimulation with PHA (5 μg/mL; Sigma Aldrich, St. Louis, MO, USA) ± IL-2 (40 U/mL; R&D Systems, Minneapolis, MN, USA) for 2.5 days, at which point cell culture supernatants were also harvested for measure of IFN-γ and IL-2. B cell proliferation was quantified using dilution of CellTrace Violet after stimulation with anti-CD40 monoclonal antibody (clone G28-5, at a concentration of 10 μg/mL, Enzo Biochem, Farmingdale, NY, USA) + IL-21 (50 ng/mL, Cell Sciences, Newburyport, MA, USA) for 3 days. For IL-21 secretion, PBMCs were stimulated with PHA for 7 days, followed by PHA + IL-23 (100 ng/mL, R&D Systems, Minneapolis, MN, USA) for 72 hours. For IL-23 secretion, patient and control PBMCs were stimulated with LPS (100 ng/mL, InvivoGen, San Diego, CA, USA) for 18 hours. For IL-12 secretion, live B-LCLs were isolated after eliminating dead cells using the Dead Cell Removal Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) per the manufacturer’s guidelines. Remaining live B-LCLs were stimulated with pDBU (10⁻⁷ M, Sigma Aldrich, St. Louis, MO, USA) for 18 hours as previously described.¹

Beaussant-Cohen S., Jaber F., Massaad M.J., Weeks S., Jones J., Alosaimi M.F., Wallace J., Al-Herz W., Geha R.S, & Chou J. (2019). Combined immunodeficiency in a patient with deficiency of c-Rel. The Journal of allergy and clinical immunology, 144(2), 606-608.e4.

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Lentivirus-mediated Knockdown of lncSnhg7 in Primary CD4+ T Cells

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Virus preparation: HEK-293T cells were transfected with the lentiviral plasmid pSMART mCMV/TurboRFP Non-Targeting Control#1 (Horizon Discovery Dharmacon, VSC6571) or a pool of pSMART mCMV/TurboRFP cloning vector (Horizon Discovery Dharmacon, V3SM11247-245870093, -245931044, -246050202) containing shRNA sequences against lncSnhg7 and lentiviral packaging plasmids, PsPax2 (Addgene, 12260) and VSV-G (Addgene, 8454), using calcium phosphate transfection. Lentivirus was collected after 48 hours and filtered (0.45 µm).
Target cell preparation and transduction: Primary CD4⁺ T cells were isolated from mice splenocytes as described above. Cells were stained with Cell Trace Violet (405/450), (Invitrogen, CellTrace™ Violet Cell Proliferation Kit, for flow cytometry, C34557). Filtered lentivirus was supplemented with LentiBOOST-P (Mayflower Bioscience, SB-P-LV-101-11) at a 1:100 dilution. Virus was added to target CD4⁺ T cells and spinoculated by centrifugation for 45 min at 500 RCF. Cells were stimulated as described above with Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation (Gibco, 1152D) and left to incubate overnight with virus for 18 h. Virus was removed, cells were resuspended in fresh media, and the T cells allowed to proliferate in the incubator for 72 h total. Cells were analyzed by flow cytometry and proliferation was modeled as described in section 2.9.

McCall J.L., Varney M.E., Rice E., Dziadowicz S.A., Hall C., Blethen K.E., Hu G., Barnett J.B, & Martinez I. (2022). Prenatal Cadmium Exposure Alters Proliferation in Mouse CD4+ T Cells via LncRNA Snhg7. Frontiers in Immunology, 12, 720635.

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Suppression of T Effector Cell Proliferation

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MACS-sorted CD4⁺ CD25⁻ T effector cells were cultured in RPMI 1640 medium in a 96-well plate at a concentration of 1 × 10⁵ cells per well. Soluble anti-CD3 (1 μg/ml; clone 2C11; BD Pharmingen) and irradiated APCs were used in a 1:2 ratio. APCs as stimulator cells were prepared from syngeneic mice by depleting the CD4⁺ T cell fraction using anti-CD4 microbeads according to the manufacturer's instructions (Miltenyi Biotec). For suppression, CD4⁺ CD25⁺ MACS-sorted A20^F/F CD4^cre+ or A20^F/F CD4^cre− T_reg cells were added at the indicated ratio to A20^F/F CD4^cre− T effector cells. Proliferation of CD4⁺ CD25⁻ T effector cells was analyzed using the CellTrace Violet Cell Proliferation Kit (Invitrogen) according to the manufacturer's instructions. The percentage of proliferating cells was analyzed after 72 h.

Fischer J.C., Otten V., Kober M., Drees C., Rosenbaum M., Schmickl M., Heidegger S., Beyaert R., van Loo G., Li X.C., Peschel C., Schmidt-Supprian M., Haas T., Spoerl S, & Poeck H. (2017). A20 Restrains Thymic Regulatory T Cell Development. Journal of immunology (Baltimore, Md. : 1950), 199(7), 2356-2365.

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Expansion of Dendritic Cell Subsets

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Female C57BL/6 mice were injected subcutaneously with melanoma cell line B16 secreting FLT3L that results in a massive expansion of all DC subsets in vivo (4 (link), 44 (link)). Seven days after inoculation, spleen and liver were harvested and pDCs were enriched by gradient centrifugation (Lymphoprep®, STEMCELL technologies) at 800 g for 20 min at 20°C without break. pDC content and number was measured by flow cytometry staining before transfer. Cell suspension was stained with carboxyfluorescein diacetate succinimidyl ester (CSFE, Molecular Probe). When indicated, pDCs were generated in vitro by incubating BM with FLT3L for 7 days. Cells were harvested and pDCs were quantified by FACS staining before labeling with CellTrace™ Violet Cell Proliferation Kit (Invitrogen™).

Stutte S., Ishikawa-Ankerhold H., Lynch L., Eickhoff S., Nasiscionyte S., Guo C., van den Heuvel D., Setzensack D., Colonna M., Maier-Begandt D., Weckbach L., Brocker T., Schulz C., Walzog B, & von Andrian U. (2022). High-Fat Diet Rapidly Modifies Trafficking, Phenotype, and Function of Plasmacytoid Dendritic Cells in Adipose Tissue. The Journal of Immunology Author Choice, 208(6), 1445-1455.

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Characterization of PDAC PDX Models

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All PDAC PDXs were obtained from Charles River Discovery Research Services Germany GmbH. PDX models were dissociated using the Tumor Dissociation Kit, human in combination with the gentleMACS™ Octo Dissociator with Heaters (both Miltenyi Biotec). Subsequently, mouse cells were depleted using the Mouse Cell Depletion Kit (Miltenyi Biotec). Resulting cell suspensions were analyzed using the MACS^® Marker Screen, human (Miltenyi Biotec) a monoclonal antibody panel containing 371 pre-titrated antibodies with nine isotype controls, or candidate antibodies selected from this panel for subsequent screening steps (Supplementary Fig. 1). To differentiate two PDX samples in one measurement, one sample was stained with the CellTrace™ Violet Cell Proliferation Kit (Invitrogen™). All samples were measured on a flow cytometer.

Schäfer D., Tomiuk S., Küster L.N., Rawashdeh W.A., Henze J., Tischler-Höhle G., Agorku D.J., Brauner J., Linnartz C., Lock D., Kaiser A., Herbel C., Eckardt D., Lamorte M., Lenhard D., Schüler J., Ströbel P., Missbach-Guentner J., Pinkert-Leetsch D., Alves F., Bosio A, & Hardt O. (2021). Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma. Nature Communications, 12, 1453.

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Flow Cytometry-Based Macrophage Phagocytosis Assay

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After maturation, macrophages were stained with 2 µM CFSE and detached using 5 mM EDTA and scraping. After washing with PBS, macrophages were re-suspended in RPMI 1640 medium supplemented with 10% FCS and 1x GlutaMax and allowed to re-attach in 96-well culture plates for 24 hours. Lymphoma cell lines (target cells) were labelled using CellTrace Violet Cell Proliferation Kit (Invitrogen) and co-cultured with macrophages with or without antibodies (10 nM) for 3 hours at a 2:1 E:T ratio. The cells were detached using 0.05% Trypsin-EDTA, transferred into multi-well plates and the assay was read out using flow cytometry. Phagocytosis was defined as the percentage of CellTrace Violet+CFSE+ cells out of all CellTrace Violet+ cells.

Patra-Kneuer M., Chang G., Xu W., Augsberger C., Grau M., Zapukhlyak M., Ilieva K., Landgraf K., Mangelberger-Eberl D., Yousefi K., Berning P., Kurz K.S., Ott G., Klener P., Khandanpour C., Horna P., Schanzer J., Steidl S., Endell J., Heitmüller C, & Lenz G. (2023). Activity of tafasitamab in combination with rituximab in subtypes of aggressive lymphoma. Frontiers in Immunology, 14, 1220558.

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Cytotoxicity Assay of Antigen-Specific T Cells

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Autologous PBMCs were labeled using the CellTrace Violet Cell Proliferation Kit (Invitrogen, Waltham, MS USA), seeded into 24 well plates and loaded with the appropriate antigen (Asp-lysate, single peptide pools or PepMix, as mentioned above). Unloaded PBMCs served as control for unspecific killing. Expanded csaATCs and seATCs were harvested, counted and seeded into 96 well plates together with unloaded or loaded PBMCs in different effector-to-target ratios (1:1, 5:1, 10:1). As a control for target cell viability, target cells were incubated in absence of T cells. Overnight co-culture supernatants were collected for subsequent multiplex analysis. Cells were washed and stained with 7-AAD and analyzed via flow cytometry using the BD FACSCanto 10c system.

Tischer-Zimmermann S., Salzer E., Bitencourt T., Frank N., Hoffmann-Freimüller C., Stemberger J., Maecker-Kolhoff B., Blasczyk R., Witt V., Fritsch G., Paster W., Lion T., Eiz-Vesper B, & Geyeregger R. (2023). Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure. Frontiers in Immunology, 14, 988947.

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Tracking Oncolytic Virus Infection in MSCs

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Cultured MSCs labeled using the CellTrace Violet Cell Proliferation Kit (Invitrogen) were infected with vMyx-EGFP/tdTr (MOI = 10) for 1 h (5% CO₂, 37°C). The vMyx-EGFP/tdTr tandem system allows expression of EGFP at both early and late infection stages (early/late promoter), while tdTr is expressed only at the late infection stage (poxvirus p11 late promoter).³⁷ (link) Unbound virus was washed away and 50 μg/mL of Ara-C solution (Ara-C is an inhibitor of poxviral DNA replication and late gene expression of MYXV) was added to evaluate the infection. After 24 h, B16-F10 melanoma cultures were stained with CellTrace Far Red reagent (Invitrogen), and, following trypsinization, melanoma cells were added to the Ara-C-treated or untreated MSC cultures (1:1 cell-to-cell ratio). The co-culture was incubated further (5% CO₂, 37°C) up to 48 h. Infection was evaluated using fluorescence microscopy (Leica) and flow cytometry (BD FACSCanto).

Jazowiecka-Rakus J., Sochanik A., Rusin A., Hadryś A., Fidyk W., Villa N., Rahman M.M., Chmielik E., Franco L.S, & McFadden G. (2020). Myxoma Virus-Loaded Mesenchymal Stem Cells in Experimental Oncolytic Therapy of Murine Pulmonary Melanoma. Molecular Therapy Oncolytics, 18, 335-350.

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Cell Proliferation Assay Protocol

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Cell proliferation assays were conducted using CellTrace™ Violet cell proliferation kit according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Briefly, cells were incubated in 5 μM of dye in PBS solution for 20 min at room temperature, washed with culture medium, seeded into 6-well plates at 0.4 × 10⁶ cells per well, and cultured for 4 days. Plates were then washed with PBS to remove nonadherent dead cells, and live adherent cells were harvested with 0.25% Trypsin/10 mM EDTA in PBS, stained for intracellular markers, and analyzed by flow cytometry.

Dawson L.E., D'Agostino L., Hakim A.A., Lackman R.D., Brown S.A., Sensenig R.B., Antonello Z.A, & Kuzin I.I. (2020). Induction of Myogenic Differentiation Improves Chemosensitivity of Chemoresistant Cells in Soft-Tissue Sarcoma Cell Lines. Sarcoma, 2020, 8647981.

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