Spleens and lymph nodes were harvested from OT-1/Rag−/− mice. Antigen-specific CD8+ OT-1 T-cells were isolated by positive selection (CD8a MicroBeads, Miltenyi Biotec, Auburn CA) and labeled with CFSE per manufacturer protocol (CellTrace™ CFSE kit, Thermo Fisher Scientific, Waltham MA). CFSE-labeled CD8+ OT-1 cells were re-suspended in 200uL PBS and transferred by retro-orbital injection (2×106 cells) on day 13 (48 hours after stereotactic RT) into congenic B6-Ly5.1/CD45.1 recipient tumor-bearing mice that had been implanted with 1.5×106 MC38-OVA cells on day 0. On day 16 (120 hours after RT, 72 hours after adoptive transfer), tumor and DLN were harvested and immune cells were isolated as above and stimulated with 2μM H-2Kb–restricted class I epitope SIINFEKL OVA257–264 (AnaSpec, Fremont CA) in the presence of GolgiStop™ and GolgiPlug™ protein transport inhibitors (BD Biosciences, San Jose CA) for intracellular cytokine staining and then analyzed by flow cytometry. As a positive control, mice were vaccinated with an attenuated Listeria monocytogenes vector engineered to express OVA (LM-OVA), (Aduro Biotech, Berkeley CA) [25 (link)]. LM-OVA was diluted in PBS to 1×107 cfu per dose (0.1 LD50), and administered by i.p. injection [21 (link)].
Celltrace cfse kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellTrace CFSE kit is a fluorescent cell labeling reagent used for tracking and monitoring cell division in vitro. It functions by labeling cellular proteins, allowing the visualization and quantification of cell proliferation.
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Lab products found in correlation
Lsrfortessa, by BD (3 mentions)
7 aad, by BD (3 mentions)
Accuri c6, by BD (2 mentions)
Golgistop, by BD (1 mentions)
Cd8a microbeads, by Miltenyi Biotec (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
αcd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Facscanto 2 flow cytometer, by Tree Star (1 mentions)
Cd49b dx5 microbeads, by Miltenyi Biotec (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Rhil 2, by Merck Group (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Flow jo version 10, by FlowJo (1 mentions)
Precision counting beads, by BioLegend (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Brdu flow cytometry kit, by BD (1 mentions)
19 protocols using celltrace cfse kit
1
Adoptive Transfer of OT-1 T-Cells into Tumor-Bearing Mice
2
Isolation and Functional Characterization of Tumor-Infiltrating CD8+ T-Cell Subsets
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CXCR5+ TIM-3–, CXCR5– TIM-3–, and CXCR5– TIM-3+, pregated as CD8+ Aqua–, were isolated from freshly minced tumors with a FACSAria cell sorter (BD Biosciences) and stained with CellTrace CFSE kit (final concentration: 2 µM; Thermo Fisher) according to the manufacturer's protocol. Subsequently, cells were plated in R10 in U-bottom 96-well plates (10,000 cells/well) and stimulated with either αCD3/CD28 Dynabeads (Life Technologies; 2 beads/cell) in combination with IL-2 and IL-15 (Peprotech; 10 ng/ml each) or IL-15 alone (50 ng/ml) for 6 and 11 d at 37°C, respectively. T cells kept in low dose of IL-15 (1 ng/ml) served as non-proliferating, negative control. Cells were then stained with mAbs (Table S1) as described above. Proliferation index depicted in Fig. 5 D was calculated as follows: Proliferation index = MFI non-proliferating fraction / MFI proliferating fraction × % cells with diluted CFSE.
3
Cell Proliferation Analysis by CFSE
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Cell proliferation was evaluated using a Cell Trace CFSE kit (C34554, Thermo Fisher Scientific). The free amino group was labeled by CFSE and dilution after cell division was analyzed by flow cytometry. The cells were seeded in a 6-well plate at 2 × 106 cells/well, cultured for 24 h and incubated with 5 μM CFSE in PBS for 15 min at 37 °C, followed by culture in the medium for 24 h. CFSE fluorescence was analyzed by a FACS Verse flow cytometer (BD Bioscience) with 488 excitation and emission filters. Fluorescence was compared with CFSE-incubated cells at corresponding time points for immediate analysis.
4
Adoptive Transfer of Labeled NK Cells
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Splenic NK cells from WT mice were isolated by magnetic bead labeling (CD49b+ DX5 microbeads) following manufacturer’s instructions (Miltenyi Biotec). Before magnetic separation, 1 x 107 cells were labeled with CellTrace CFSE kit (Thermo) according to the manufacturer’s protocol. Labeled NK cells (1 x 106 cells/mouse) were adoptively transferred intravenously into ifnar1-/- mice, which were infected intranasally with 3 x 105K. pneumoniae 52.145 CFU. 24 h post infection mice were euthanized and bacterial loads in lungs determined. Intracellular IFN-γ was detected using anti IFN-γ (XMG1.2). Cells were analyzed using a FACSCantoII flow cytometer and FlowJo software (Tree Star).
5
Visualizing TAMs Phagocytosis of A549 Cells
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The phagocytosis of TAMs was detected by a Cell Trace CFSE kit (Thermo Fisher, USA). A549 cells were labeled with Cell Trance Yellow reagent according to the manufacturer's instructions, and 1 × 105 TAMs tagged with GFP were inoculated into 6-well plates. The cells were incubated in RPMI complete medium for 2 h and then mixed with labeled A549 cells. The cells were observed and photographed by fluorescence microscopy, and phagocytosis was detected by flow cytometry.
6
Evaluating Trophoblast Invasion Using HESC Co-Culture
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HESC cells decidualized and non-decidualized were grown to confluence in 24 wells plate (Greiner Bio-One, Kremsmünster, Austria).The inhibitor STF-083010 was added for 4 h prior to the invasion assay. Swan-71 spheroids were morphologically selected, stained with CFSE (CellTrace CFSE Kit, ThermoFisher Scientific, MA, USA) according providers instructions and ten spheroids per well were transferred using a transfer pipette and a dissecting microscope. The co-culture was maintained with DMEM supplemented with 10% FBS without differentiation stimuli nor STF-083010. All co-cultures were monitored using a fluorescence microscope (Olympus Lifesciences, USA) and microphotographs were taken at 48 h, as we previously determined to be the optimal invasion time18 . Invasion index was analyzed as morphological change and calculated as “1-minor_axis/mayor_axis” of an ellipse surrounding the BLS as previously reported using ImageJ software (NIH, USA)18 . For each assay, the average of the invasion index of all the BLS on each well was considered as one single sample and used for the statistical analysis.
7
T Cell Proliferation Monitoring by CFSE
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Proliferation was monitored as a function of CFSE dilution. CD3+ T cells were stained with 1μM CFDA-SE (Cell Trace CFSE kit, ThermoFisher) for 3 min and washed twice with PBS 2% FCS. T cells (1x106) were subsequently plated into anti-CD3/anti-CD28 coated wells (clones OKT3 and 9.3 respectively, BioXCell) or stimulated with PHA (1μg/ml-Sigma) and rhIL2 (100IU/ml-Proleukin). Proliferation was evaluated at day 5 following staining with anti-CD4 APC (clone 13B8.2) and anti-CD8 PeCy7 (clone SFC121Thy2D3) mAbs and data acquired on a FACSCanto II Flow Cytometer (Becton Dickinson). Analyses were performed using FlowJo Software (v10–10.6.2).
8
HESC and Swan-71 spheroids co-culture
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HESC cells decidualized in presence of MPA-db-cAMP or VIP andnon-decidualized were grown to confluence in 24 wells-plate (Greiner Bio-One, Kremsmünster, Austria). Swan-71 spheroids were stained with CFSE (CellTrace CFSE Kit, ThermoFisher Scientific, MA, USA) according providers instructions and then (ten spheroids per well) transferred using a transfer pipette and a dissecting microscope. They were co-cultured with confluent HESC and maintained in DMEM supplemented with 10% FBS. All co-cultures were monitored using an Olympus microscope (Olympus Lifesciences, USA) and ImageJ software (NIH, USA). Invasion index was analysed as morphological change and calculated as "1-minor_axis/mayor_axis" of an ellipse surrounding the BLS as shown on figure 3C. For each assay, the average of the invasion index of all the BLS on each well was considered as one single sample and used for the statistical analysis.
9
Apoptosis and Proliferation Analysis
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Cells were harvested 48 h after transfection, and then digested by 0.25% trypsin without EDTA and collected in 1.5 ml Eppendorf tubes. Cells were washed twice using washing buffer, and then incubated with Annexin V-FITC and propidium iodide (cat. no. 40302ES20; Yeasen, Shanghai, China) in the dark at 25°C for 20 min. Binding buffer was added to each tube and apoptosis analyzed within 1 h. The apoptosis rate is derived from the addition of right upper quadrant and right lower quadrant together.
Cell proliferation was measured using carboxyfluorescein succinimidyl ester (CFSE) (29 (link)). Cells were labeled with CellTrace ™ CFSE kit (C34554; Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 37°C and then washed twice with phosphate-buffered saline. The cells were incubated for at ≥10 min before analysis to allow the CellTrace™ reagent to undergo acetate hydrolysis. Fluorescence was measured using a flow cytometer and Flow Jo version 10.0 software (FlowJo LLC, Ashland, OR, USA).
Cell proliferation was measured using carboxyfluorescein succinimidyl ester (CFSE) (29 (link)). Cells were labeled with CellTrace ™ CFSE kit (C34554; Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 37°C and then washed twice with phosphate-buffered saline. The cells were incubated for at ≥10 min before analysis to allow the CellTrace™ reagent to undergo acetate hydrolysis. Fluorescence was measured using a flow cytometer and Flow Jo version 10.0 software (FlowJo LLC, Ashland, OR, USA).
10
EpCAM BiTE Modulates T-cell Proliferation
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To study T‐cell proliferation, 100,000 CFSE‐labelled (CellTrace CFSE kit, Invitrogen, #C34554) CD3+ T cells were incubated with 20,000 DLD cells in 96‐well plate format, with 2 ng/μl EpCAM or control BiTE. Five days after co‐culture, cells were stained for CD3, CD4 or CD8 and CFSE fluorescence of viable CD3+ T cells was measured by flow cytometry, with total cell number normalised using precision counting beads (5,000/well, Biolegend, #424902). Fluorescence data were analysed and modelled using the proliferation function of FlowJo v7.6.5 software. Data are presented as the percentage of original cells that entered a proliferation cycle (%divided) or the average number of cell divisions that a cell in the original population has undergone (Division Index).
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