A general description of the in vitro adhesion assay of cancerous cells on NETs is described in (17 ). Briefly, isolated NETs were left to adhere overnight at 4°C. The next day, nonadherent NETs were aspirated, and wells were blocked with 1% BSA for 1 h at RT. After blocking, 200 μg/ml anti-CEACAM1 mAb (5F4) or 200 μg/ml mouse IgG isotype control (34B1, from Dr. Blumberg) were added to some wells and incubated for 1 h at 37°C and 5% CO2. After washing, 2 × 104 HT-29 or A549 cells were stained with CFSE dye for 10 min (Life Technologies) and then washed and added to the NET monolayers and incubated for 90 min at 37°C, 5% CO2. Cells were subsequently gently aspirated, washed once, and fixed in 4% paraformaldehyde. In some experiments, 1000 U of DNase I were added to NET–cell mixtures 10 min prior to fixation and quantification of adhesion. Adhesion was quantified as the number of cells in 4 random high-power fields (hpf) at 20× using a Nikon TE300 microscope (Nikon, Mississauga ON).
Cfse dye
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
CFSE dye is a fluorescent labeling agent used for tracking and monitoring cell division. It binds to cellular proteins, allowing the fluorescent labeling of cells. The dye is partitioned equally between daughter cells during cell division, enabling the visualization and quantification of cell proliferation.
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Lab products found in correlation
Exoquick exosome isolation reagent, by System Biosciences (3 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Facsaria 2 cell sorter, by BD (2 mentions)
Transwell plate, by Corning (2 mentions)
Te300 microscope, by Nikon (1 mentions)
96 well flat bottomed microplates, by Greiner (1 mentions)
Anti cd3 apc, by BioLegend (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Pe anti mouse cd8, by BioLegend (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by BD (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
Annexin 5 and 7 aad, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Celltrace violet ctv dye, by Thermo Fisher Scientific (1 mentions)
96 well round bottom plate, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by STEMCELL (1 mentions)
T cell enrichment kit, by STEMCELL (1 mentions)
57 protocols using cfse dye
1
In vitro Adhesion Assay of Cancer Cells on NETs
2
CFSE-Labeled T Cell Activation
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Isolated effector T cells (CD3+ CCR7−) were labeled with the CFSE dye (Life Technologies) before stimulation using the Dyna beads Human T-activator CD3/CD28 (Thermo Fisher Scientific) according to the manufacturer’s instruction and our protocols [53 , 54 (link)]. After 72 h cells were stained and analyzed.
3
Allogenic PBMC Proliferation Assay
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Mo‐DCs (stimulator cells) were inactivated by γ‐radiation at 30 GY, 100% and seeded into 96‐well flat‐bottomed microplates (Greiner Bio‐One, Frickenhausen, Germany) at concentrations of 2 × 104 cells/well. A total of 2 × 105 responding cells from unmatched allogenic 5(6)‐carboxyfluorescein N‐hydroxysuccinimidyl ester (CFSE)‐labeled PBMCs from healthy volunteers were added. Phytohemagglutinin (PHA) stimulation (3 µg/ml; Sigma‐Aldrich) of CFSE‐labeled PBMCs and native CFSE‐labeled PBMCs served as positive and negative proliferation controls, respectively. Distinct generations of proliferating cells were monitored by CFSE dye (Life Technologies/Thermo Fisher Scientific, Darmstadt, Germany) dilution. After co‐culture for 6 days, fluorescence activated cell sorter (FACS) analysis of recovered lymphocytes was performed and CD4‐PE antibody (BD Bioscience) served to identify CD4+ T cells. All cell counts were scaled to the 0–100% range using GraphPad Prism’s ‘normalize’ function; 0% was defined as the value of PBMC without PHA and 100% as the value of PHA‐stimulated PBMC. GraphPad Prism version 8.1.0 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com ).
4
CFSE-based Proliferation and Cytokine Assay for mesoCAR T Cells
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Target cells were treated with 50 μg/ml of mitomycin C (Sigma Aldrich) for 30 minutes at 37°C and extensively washed (28 (link)). To track cell proliferation using CFSE dye (Life Technologies), mesoCAR T cells and untransduced T cells (1.2 × 107/ml) were stained with 5 μM CFSE at room temperature for 8 minutes. The reaction was terminated by adding an equal amount of FBS. After washing three times with complete RPMI 1640 medium, CFSE-labeled cells (0.2 × 106/well) were cocultured at a 1:1 ratio with either target cells or cultured in media (without target cells) in the absence of exogenous IL‐2 in 48‐well plates, with a final volume of 800 μl/well. The supernatant was harvested 24 hours after plating and stored at −20°C until subsequent cytokine analysis by enzyme‐linked immunosorbent assay (ELISA) to quantify IFN‐γ and IL‐2. After 72 hours, cells were stained with anti‐CD3‐APC (Clone: UCHT1, BioLegend), and CFSE dilution of CD3+ cells, as a measure of proliferation, was determined by flow cytometry, as previously described (29 (link)).
5
T Cell Proliferation Assay
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Isolated CD3+ T cells were labeled with the CFSE dye (Life Technologies) and then stimulated with the Dyna beads Human T-activator CD3/CD28 (Thermo Fisher Scientific) according to the manufacturer’s protocol and analyzed after 72 hours.
6
Activation of Memory CD8 T Cells
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Sorted naïve and memory CD8 T cell subsets were labeled with cell trace violet (CTV) dye at 2.5 µM and carboxyfluorescein succinimidyl ester (CFSE) dye at 1 µM respectively (Life Technologies). To generate polyclonal activated memory CD8 T cells, each memory CFSE-labeled subset was stimulated in vitro using Dynabeads Human T-Activator anti-CD3/CD28 coated magnetic beads [1:1 ratio] (Gibco) in a 96-well round-bottom plate for 18 h, followed by bead removal using a plate magnet (Thermo-Fisher Scientific).
7
CFSE-Based RA-FLS Proliferation Assay
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FLS proliferation stimulated by RA serum or leptin (100 ng/mL) was assessed by CFSE dye (eBioscience, USA). CFSE is a fluorescent probe used to label live cells that were taken to daughter cells in the same way during division. Single-cell suspension of RA-FLS was tagged with CFSE (10 μM) for 10 min in the dark at 37 °C. Wash cells with culture media to remove unincorporated CFSE. Flow cytometry was used to analyze RA-FLS division tracking.
8
CFSE-Based CD8+ T Cell Proliferation Assay
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CD8+ T cells were labelled with CFSE dye (carboxyfluorescein succinimidyl ester, 1 µl ml−1, eBioscience, CA, USA) at 37°C for 15 min and RPMI-1640 with 10% FBS (5 min) was added to terminate the reaction. Antigens (freeze–thawed 4T1 tumour lysate) were then added at a concentration of 50 µg ml−1 to simulate T-cell proliferation. CD8+ T cells were collected on day 3 following the addition of lysate and the proliferation rate was assessed by the progressive dilution of CFSE dye. CD8 and IgG2a K on CFSE-labelled T cells were stained using PE anti-mouse CD8 (BioLegend, CA, USA) and PE Mouse IgG2a K Isotype Control antibodies before flow cytometry using CytoFLEXS.
9
F8CAR-Treg Suppression Assay
10
Quantifying Chemotherapy-Induced ROS
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Reactive oxygen species (ROS) were measured using the redox-sensitive dye CM-H2DCFDA (Invitrogen, Grand Island, NY). To control for altered dye retention due to cell division, another aliquot of cells was simultaneously labeled with carboxyfluorescein succinimidyl ester (CFSE) dye (eBioscience, San Diego, CA) [62 (link)]. Briefly, cells were labeled with CFSE for 24 hours or with CM-H2DCFDA for 30 minutes and treated with chemotherapy drug and/or AZD1208 or DMSO control. Cells were harvested at 8, 24, 48 and 72 hours and fluorescence was measured on a FACSCanto II and analyzed using FlowJo. Divergence in the CFSE and CM-H2DCFDA curves at serial time points indicates altered ROS generation.
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