Lung cells were isolated and enriched for CD90.2 cells as described above, followed by labeling with CellTrace Violet (Cat# C34557; Thermo Fisher Scientific) according to manufacturer’s instructions and subsequently stained with antibodies. After 3-day culture of sort-purified ILCs with 20ng/ml IL-7 either alone or in combination with 200ng/ml IL-33, 100pM CGRP or 100nM CGRP followed by staining, expression of CellTrace Violet in live (7AAD-) ILCs was analyzed on a BD LSRFortessa (BD Biosciences).
Celltrace violet
Manufactured by BD
Sourced in United States
CellTrace Violet is a fluorescent cell staining dye used for labeling and tracking cells. It binds to cellular proteins, allowing visualization and analysis of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (25 mentions)
Facscanto 2, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Facsaria 2, by BD (2 mentions)
Poly 1 c, by InvivoGen (2 mentions)
Pandc separation kit, by STEMCELL (2 mentions)
Facsaria, by BD (2 mentions)
Celltrace violet dye, by Thermo Fisher Scientific (2 mentions)
No f6765, by Merck Group (2 mentions)
Tc20 automated cell counter, by Bio-Rad (2 mentions)
Gallios instrument, by Beckman Coulter (1 mentions)
Dynabeads human t activator, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa sorp flow cytometer, by BD (1 mentions)
Anti cd3 okt3, by Thermo Fisher Scientific (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
X vivo 15 serum free medium, by Lonza (1 mentions)
Anti cd3 145 2c11, by BioXCell (1 mentions)
Anti cd28 37, by BioXCell (1 mentions)
Anti il 4 11b11, by BioXCell (1 mentions)
21 protocols using celltrace violet
1
Isolating and Labeling Lung ILCs
2
Cas9 Nucleofection and CellTrace Violet Labeling
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After isolation and stimulation, primary human T cells were nucleofected with Cas9 RNPs identical to what was described earlier. Immediately after nucleofection recovery, cells were pelleted and resuspended in 5 μM CellTrace Violet (Invitrogen). Cells were incubated in CellTrace Violet for 20 min at 37° C, prior to diluting in 4x volume of complete X-Vivo 15 medium to absorb unbound dye and incubating again for 5 min at 37° C. Cells were pelleted and resuspended in complete X-Vivo 15 with 300 U/mL IL-2. T cells were passaged every other day to refresh medium and maintain a density of ~0.5–1×106 cells/mL. Cells were sorted on a BD FACSAria II to obtain the approximate bottom and top quartile of cells according to CellTrace Violet signal.
3
T-cell Proliferation Assay with MSCs
4
T Reg Suppression Assay Protocol
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Patient samples collected on C3D21 with a Treg content of 15–40% of the CD4+ population were used in Treg suppression assays ex vivo. PBMCs collected from healthy donors served as control. Cells were stained with anti-human monoclonal antibodies as described above. Tregs (CD4+CD14−CD25hiCD127low) and conventional CD4+ T cells (Tcons; CD4+CD14−CD25lowCD127hi) were sorted on a 3-laser BD FACSAriaIII flow cytometer (405, 488 and 640 nm; BD Biosciences). The gating strategy is shown in Supplementary Fig. 2. The sorted Tcons were stained with CellTrace™ violet (Life Technologies) and 35,000 cells per well were seeded together with 2 µg/ml soluble anti-CD28, in X-VIVO™ 15 serum-free medium (Lonza Group Ltd, Basel, Switzerland) to a 384-well plate coated with anti-CD3 (OKT3; eBioscience). An equal number of Tregs (35,000/well) was added to half of the wells. After 4–5 days of culture the proliferation of Tcons was determined by measuring the intensity of the CellTrace™ violet staining on an LSRFortessa SORP flow cytometer (BD Biosciences).
5
T Cell Activation and Differentiation
6
Splenocyte Activation Assay with Kasugamycin and PolyI:C
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Single cell splenocytes were plated at the density of 5×106 cells/ml isolated and treated with kasugamycin (2mg/ml or 0.02mg/ml) and PolyI:C (High Molecular Weight, Invivogen,CA) (2μg/ml or 0.02μg/ml) for 6 hours. Mixtures of PolyI:C and kasugamycin were incubated together for 30 minutes at 37°C before addition to cells. In Supplementary Fig. 13 , splenocytes were treated for 12 hours, washed 5 times, stained with cell trace violet (Thermo Fisher, CA), and incubated with splenic DCs isolated using PanDC separation kit (Stemcell Technologies, MA). After 6 hour incubation, cell trace violet dim and negative cells were sorted using an FACSAria (BD, NJ) into RLT buffer for RNA extraction. All in-vitro experiments were conducted with a minimum of three replicates and repeated twice.
7
Tetanus Toxoid Immune Complex Assay
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Tetanus toxoid immune complexes (TT-IC) were generated by incubation of tetanus toxoid (TT; Statens Serum Institut, Denmark) with HyperTet S/D hyper-immune IgG serum (Grifols, USA). ICs were prepared in 96-well round bottom cell culture plates as 2x (100 µl) solutions in complete RPMI-1640 (10% FCS) medium. Briefly, TT (2.0 µg/ml) was incubated with HyperTet (2.0 mg/ml) over-night at 4 °C. PBMCs were then labelled with CellTrace Violet (ThermoFisher) according to the manufacturer’s instructions. Labelled PBMCs (300,000/well) were subsequently added in the same volume to the 2x TT-IC solution to give rise to a final concentration of 1.0 µg/ml of TT complexed in 1.0 mg/ml of HyperTet. Other assay conditions included TT only (1.0 µg/ml), or TT-ICs with hexameric-Fc (50 µg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 µg/ml per antibody). Cells were incubated for 6d at 37 °C. Subsequently, T cell division was assessed through flow cytometry (BD Fortessa) by determining the percentage of T cells having undergone CellTrace Violet dye dilution compared to unchallenged control conditions gating on T cells using a CD3-APCH7 antibody (BD). To aid comparison between independent experiments, T cell division was normalised taking into account minimal and maximal responses within individual experiments.
8
Regulatory T Cell Suppression Assay
9
Cell Proliferation Assays and E2-Dependent Proliferation
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For trypan blue exclusion assays, cells were trypsinized and mixed with trypan blue solution. Viable cells were determined based on dye exclusion and were counted using a TC20 Automated Cell Counter (Bio-Rad). CellTrace Violet proliferation assays were performed by staining cells with CellTrace Violet dye (Thermo Fisher) according to the manufacturer’s instructions and culturing cells for a total of seven days. A portion of cells were fixed with 10% formalin for 20 minutes on days 4, 7, and 10, washed with PBS, and stored in PBS at 4°C until analysis. CellTrace Violet fluorescence intensity was analyzed in the VUMC Flow Cytometry Shared Resource using the 5-laser BD LSRII. Datasets were analyzed using FlowJo software (FlowJo, LLC). For E2-dependent proliferation assays, cells were grown in charcoal-stripped FBS-containing (cs-FBS) media (Millipore Sigma, Catalog No. F6765) for 1.5 weeks prior to initiation of experiments to ensure estrogen deprivation. Cells were cultured in cs-FBS media in the presence or absence of E2 (10nM; Millipore Sigma, Catalog No. E2758) for a total of 3 days. Each day, proliferation and viability was assessed by trypan blue exclusion.
10
T Cell Proliferation Assay with LrS235 and LrGusA
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Peripheral blood MNC were loaded with 5 µM CellTrace Violet (Invitrogen) according to the manufacturer’s instructions (58 (link)) and incubated for 30 min with sterile-filtered supernatants from LrS235 or LrGusA, buffered to pH 7.4 and diluted 1/10 in tissue culture media, before the addition of anti-CD3 antibodies to stimulate T lymphocytes (clone OKT3, 1 ng/ml, 037-85, Thermo Fisher). Seven days later, cells were stained with anti-CD3 antibodies conjugated to phycoerythrin (BioLegend 300308, lot B209105) and anti-CCR7 antibodies conjugated to fluorescein (R&D Systems, FAB197F, lot LEU1615081), and the dilution of CellTrace Violet in CD3+CCR7− TEM cells and CD3+CCR7+ naive/TCM cells was measured by flow cytometry on a BD FACSCantoII as quantification of cell proliferation. Data were analyzed with FlowJo.
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