PNF from mice used to prepare BMDM were collected and dissocated as described for flow cytometry, and 5 × 106 cells from each tumor labled with either 0.1 μM of CFSE Cell Division Tracker Kit (BioLegend) or CellTracker Violet BMQC Dye (5 μM; Thermo Fisher Scientific) for 15 min on ice. Cells were then washed twice, combined, and cultured in ultra-low binding round bottom plates for 8 h at 37°C and 5% CO2 in OPTI-MEM plus 1% PenStrep without added serum. After incubation, cells were removed and flow cytometry was used.
Celltracker violet bmqc dye
Manufactured by Thermo Fisher Scientific
CellTracker Violet BMQC dye is a fluorescent cell-permeant dye that can be used to label and track live cells. It is designed to passively diffuse into cells and become covalently bound, allowing for long-term labeling of cells.
Automatically generated - may contain errors
Lab products found in correlation
Dynabead, by Thermo Fisher Scientific (2 mentions)
7 aad, by BD (2 mentions)
Cfse cell division tracker kit, by BioLegend (1 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (1 mentions)
Sh800s cell sorter, by Sony (1 mentions)
Diva software, by BD (1 mentions)
Rm4 5, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd86 pe cy7, by BD (1 mentions)
Percp5, by BioLegend (1 mentions)
Facsaria 2, by BD (1 mentions)
10 protocols using celltracker violet bmqc dye
1
CFSE-based T Cell Division Assay
2
Leishmania Infection of Macrophages and Neutrophils
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L. (L.) amazonensis strain (MHOM/BR/73M2269) [26 (link)] that constitutively expresses GFP and two different L. infantum isolates from VL patients (MHOM/BR/2009/LVHSE17 as isolate 1 and MHOM/BR/2010/LVHSE49 as isolate 2) were used [27 (link)]. L. amazonensis-GFP was constructed by incorporation of the GFP gene into 18s ribosomal RNA by homologous recombination using pSSU vector, as described by Misslitz et al. (2000) [28 (link)]. L. infantum isolates were stained with CellTracker Violet BMQC dye (Thermo Fisher) as previously described [29 (link)]. The parasites were cultured axenically in Schneider's Drosophila medium (Thermo Fisher) plus 10% FBS prior to infection of cells. Infection of human macrophages was performed at a ratio of either 10 stationary-phase L. amazonensis-GFP promastigotes or 5 L. infantum per macrophage. Extracellular parasites were removed 2 hours later by washing. After 24h, the cells were then stained and subjected to flow cytometry. Neutrophils were infected at a ratio of 5 L. amazonensis parasites per neutrophil for 3h prior to staining and flow cytometry. Infection with Mycobaterium. bovis BCG (Fundação Ataulpho de Paiva) was performed at a ratio of 2 mycobacteria per macrophage. BCG were also stained with CellTracker Violet BMQC dye, following the same protocol used for Leishmania.
3
Tracking Regulatory T Cell Migration
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Live TCRβ+CD4+FoxP3eGFP+ Treg were isolated from SKG.FoxP3eGFP mice using a Sony SH800S Cell Sorter. Sorted Treg were expanded on Dynabeads (Invitrogen, 11452D, lot: 00911146) in complete RPMI supplemented with 10 ng mL−1 IL‐2 for 5 days according to manufacturer's instructions for Dynabead use. After 5 days, Treg were washed with PBS and stained for 30 min at 37 °C with CellTracker Violet BMQC dye (ThermoFisher, Cat No. C10094, lot: 2451243). For staining, cells were washed with sterile PBS and resuspended at a concentration of 106 cells mL−1 in a solution of PBS supplemented with CellTracker Violet BMQC dye, prepared by adding 2 µL of stock dye per 1 mL of PBS. Cells were then washed twice to remove residual dye and injected into a single ankle of either healthy BALB/cJ or SKG mice with established arthritis at 5 × 105 Treg suspended in 20 µL of PBS per mouse. Mice were sacrificed 3 days post‐injection and the spleens, draining lymph nodes (inguinal and popliteal), contralateral lymph nodes (inguinal and popliteal), injected ankle, and uninjected ankle were all harvested and analyzed using flow cytometry. Mice that did not receive any injection were used as a negative control for analysis.
4
NK Cell Cytotoxicity Assay with K562 Cells
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NK cells and K562 cells were labeled with 0.36 μM CFSE or with 5 μM CellTracker Violet BMQC dye (Thermo Fisher Scientific) following the manufacturer’s instructions. In addition, NK cells were stained with APC-labeled anti-CXCR2 or anti-NGFR antibodies and, in some experiments, subsequently pre-incubated with 10 mg/ml anti-CD11a for 20 min at 4 °C. Next, NK cells (1 × 105) and K562 cells (1 × 105) were mixed at an effector-to-target ratio of 1:1 in a final volume of 200 μL of X-Vivo 20 medium with 10% human AB serum, centrifuged at 4 °C for 1 min at 20 x g, and incubated in a 37 °C water bath for 10 min. Reactions were stopped by adding 0.5% paraformaldehyde. Conjugate formation was analyzed by flow cytometry, and the percentage of NK cells in conjugates was calculated as the ratio of double positive events to total effector cell events.
5
T Cell Proliferation Assay
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Splenocytes were subjected to negative selection using magnetic beads (EasySep™ Mouse T Cell Enrichment Kit). Subsequently, CD3+ splenocytes were stained with CellTracker™ Violet BMQC Dye (Thermo Fisher) for 20 min in 37 °C and washed two times. Next, CD11b+ cells and T cells were seeded onto previously coated with anti-CD3 antibody (145-2C11, eBioscience) 96-well plate in 2:1 ratio. Then, cells were stimulated with anti-CD28 (145-2C11, eBioscience) for three consecutive days. Subsequently, T cells were stained and proliferation of CD8+ (53-6.7, eBioscience) and CD4+ (RM4-5, eBioscience) cells was evaluated with FACSCanto II using Diva software (Becton Dickinson, Franklin Lakes, New Jersey, USA).
6
Metformin Enhances NK Cell Cytotoxicity
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Target cells were incubated with 2 mm Metformin for 3 days. After washing, cells were suspended in RPMI medium 10% FBS and 100.00 cells per well (96 well round (U) bottom plate) were incubated overnight in the presence or absence of e-NK cells at a 1: 1 effector: target (E: T) ratio. Previously fresh or frozen (stored in liquid nitrogen) NK cells were labeled with 3 µm of CellTracker™ Violet BMQC Dye (Life Technologies). Subsequently, viability was analyzed in the violet fluorescence negative target cell population by flow cytometry (Galllios Beckman Coulter) using 7AAD (BD Biosciencies)24 .
7
NK Cell-Mediated Cytotoxicity Assay
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This was performed as previously described (14 (link), 15 (link)). NK cells were labeled with 3 μM of CellTracker™ Violet BMQC Dye (Life Technologies) and incubated overnight with target cells at different E:T ratios. Subsequently, phosphatidylserine (PS) translocation and membrane damage were analyzed in the violet fluorescence-negative target cell population by flow cytometry using Annexin V-FITC (Immunostep) and 7AAD (BD Biosciences) or propidium iodide (PI) as previously described (16 (link), 17 (link)). We consider all cells positive for annexin-V and/or PI (or 7-ADD) as dead (or dying).
8
NK Cell-Mediated Cytotoxicity Assay
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Fresh or frozen (stored in liquid nitrogen) NK cells were labeled with 3 µM of CellTracker™ Violet BMQC Dye (Life Technologies) and incubated overnight with target cells at different E:T ratios. Subsequently, viability was analyzed in the violet fluorescence negative target cell population by flow cytometry using 7AAD (BD Biosciencies)31 .
For certain experiments, effector cells were previously incubated with anti-TRAIL mAb RIK2 (2.5 µg/mL), recombinant human Fas-Fc chimera (10 ng/mL) or calcium chelator EGTA (1 mM) for 1 h at 37 °C, before facing target cells.
For certain experiments, effector cells were previously incubated with anti-TRAIL mAb RIK2 (2.5 µg/mL), recombinant human Fas-Fc chimera (10 ng/mL) or calcium chelator EGTA (1 mM) for 1 h at 37 °C, before facing target cells.
9
Tracking OT-I T Cell Migration
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OT-I CD8+ T cells in suspension were stained with 2.5 μM Cell Tracker Violet BMQC Dye (C10094, Invitrogen) for 30 min. CM from tumor cells (0.75 mL of medium containing 5% FBS) were plated in the lower wells of a chamber assay plate, and OT-I cells (2.5 × 105 cells in 0.1 mL of medium containing 1% FBS) were placed in the upper wells of the insert. After co-incubation for 1, 2, 4, or 6 h, cells were collected from the bottom wells and subjected to flow cytometry to identify BMQC+ cells70 (link).
10
Phagocytosis Assay for Dendritic Cell-Mediated Killing of NSCLC Cells
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After in vitro generation of DCs, NCI-H1975, A549 and NCI-H1650 cells were labelled with PKH67, a green fluorescent membrane dye (Sigma Aldrich, MIDI67), prior to plating them out on day 3. NSCLC cells were treated with chemotherapy on day 4. On day 5, immature DCs were stained with cytoplasmic violet-fluorescent CellTracker Violet BMQC dye (Invitrogen, C10094) and effector (E) and target (T) cells were placed in coculture at a 1:1 (E:T) ratio. Supernatant (SN) was stored (−20 °C), cells were collected and immediately used for flowcytometric detection of DC maturation markers and phagocytosis on day 7. Cells were stained with CD80-PerCP5.5 (Biolegend, 400150) and CD86-PE-Cy7 (BD Biosciences, 557872) to assess DC maturation (Violet+ population). Isotype controls (PerCP5.5, Biolegend, 305232; PE-Cy7, BD Biosciences, 557872) were included to subtract aspecific signals from measured fluorescence intensity. Phagocytosis of NSCLC cells was assessed by gating on the PKH67+Violet+ population, as previously described [35 (link)]. Acquisition was performed on a FACSAria II (BD Biosciences). Data analysis was performed using FlowJo v10.1 software (TreeStar).
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