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7 protocols using carboxyfluorescein succinimidyl ester (cfse)

1

Neutrophil Adhesion Assay: ICAM-1 and CXCL1

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Neutrophils obtained after 4-day differentiation (“in vitro-derived”) were washed and labeled using CFSE (BioLegend). Murine bone marrow neutrophils were isolated by negative selection (StemCell Technologies) and immediately labeled alongside in vitro-derived neutrophils using CFSE. 96-well plates were coated for 1 hour at room temperature or overnight at 4 °C with 2.5, 5, or 7.5 µg/mL ICAM-1 and/or 2.5 µg/mL CXCL1 in phosphate buffered saline (PBS) and then blocked with 1% casein (ThermoFisher) or 0.5% polyvinylpyrrolidone (Sigma Aldrich) in PBS for 2 hours at room temperature. Neutrophils were loaded into the 96-well plate at 0.5 × 106 neutrophils per well in Hank’s balanced salt solution containing Ca2+ and Mg2+ (HBSS++), and then incubated at 37 °C for 35 or 65 minutes. Following incubation, neutrophils were quantified by a plate reader for fluorescence intensity (CFSE), before and after sequential gentle washes with HBSS++. The number of adherent neutrophils was inspected visually by light microscopy to corroborate with plate reader signal intensity. Each group was replicated in three to six wells per independent experiment.
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2

Murine Dendritic Cells Regulate CD4+ T Cell Activation

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Murine
bone-marrow-derived dendritic cells (BMDCs) (5 × 105 cells/mL) from BALB/c mice were pretreated with the vehicle
or broad Ca2+ channel blocker (SKF96365, 20 μM) and
treated with vehicle 2D216 or its derivatives (5 μM)
in combination with MPLA (100 ng/mL) for 24 h. BMDCs were loaded with
the OVA protein (10 μg/mL) for 4 h, washed twice, and co-cultured
with the same number of CFSE-labeled CD4 T cells from spleens of sex-matched
DO11.10 TCR transgenic mice for 72 h. CD4+ T cells were
isolated from spleens of DO11.10 TCR transgenic mice using an EasySep
Mouse CD4 T cell isolation kit (STEMCELL Technologies, Vancouver,
Canada) and labeled with CFSE (4 μM, Molecular Probe, Eugene,
OR, United States). Supernatants were assayed for IFN-γ and
IL-2 by ELISA, and cell suspensions were subjected to fluorescence-activated
cell sorting (FACS) analysis of CFSE dilution of DO11.10 CD4 T cells.
T cell division was analyzed by a MACSQuant Analyzer 10 (Miltenyi
Biotec, Bergisch Gladbach, Germany) using AF647-conjugated anti-DO11.10
TCR antibodies (eBioscience, San Diego, CA, United States). The %
divided, the percent of the live CFSE-labeled CD4+ T cells
that entered division, was calculated using FlowJo (version 10.6.1,
Becton Dickinson, Ashland, OR, United States).
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3

CFSE-Based Lymphocyte Proliferation Assay

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The carboxyfluorescein diacetate succinimidyl ester (CFSE, Biolegend, USA) was used to label the proliferation of the PBMCs. The cells were resuspended with 5 μM CFSE working fluid and incubated in a carbon dioxide cell incubator for 20 min. Next, a five-time volume of the RPIM1640 medium containing 10% fetal bovine serum was added to terminate the reaction, and then washed three times by RPIM1640 medium. After labeling with CFSE, the cells were incubated with human CD3/CD28 T cell activator (Stem Cell, Canada) at a dose of 25 µL/mL and IL-2 (Pepro Tech, USA) at a dose of 50 ng/mL for up to 3 d, and then analyzed by fluorescence-activated cell sorting (FACS).
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4

CFSE-Labeled T Cell Proliferation Assay

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PBMC were thawed and labeled with carboxyfluorescein succinimidyl ester (CFSE, Invitrogen). T cells were isolated by negative selection with the EasySep Human T Cell Enrichment Kit (STEMCELL Technologies) from CFSE-labeled PBMC. The purities of CD3+ T cells (as determined by flow cytometry) were greater than 98% after enrichment. Frozen CD B cells were thawed and counted. Both T and CD B cells were suspended well before co-culture.
Equal numbers (1 × 104 each) of T and CD B cells per well were co-cultured in 96-well U-bottom plates (Fisher Scientific) in 100 µl per well of R5 medium without exogenous cytokines. For alloreactive T-cell proliferation studies, the plates were incubated at 37°C in a 5% CO2- humidified incubator for 5 days. For microbial antigen-specific T-cell proliferation studies, the plates were incubated for 7 days. Tetanus toxoid from Clostridium tetani (List Biological Laboratories), recombinant influenza HA (H3 A/Wisconsin/67/2005, kindly provided by S.C. Harrison), and recombinant B. anthracis PA (BEI Resources) were used in antigen-specific T-cell proliferation studies. T cells were treated with equal numbers of anti-CD3/CD28 Dynabeads® (Invitrogen) as positive controls in both alloreactive and microbial antigen-specific T-cell proliferation studies.
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5

Neutrophil-T Cell Coculture Assay

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Before coculture with T cells, neutrophils were washed three times with PBS. In a 3-day incubation, bead-purified peripheral CD3+ T cells (1 × 105 cells/well in 96-well plates) were labeled with carboxy fluorescein succinimidyl ester (CFSE, Stem Cell Technology) and cocultured with neutrophils at a 1:1 ratio in 100 μl RPMI-1640 medium containing anti-CD3 (8 μg/ml) and anti-CD28 (5 μg/ml) antibodies (eBioscience). In a 1-day coculture system, neutrophils were cocultured with bead-purified CD3+ T cells at a 1:1 ratio in 100 μl RPMI-1640 medium containing anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) antibodies (eBioscience). After 1-day incubation, the cells were harvested for flow cytometric analysis of CD69 and interferon (IFN)-γ. For blockade study, the treated neutrophils were cocultured with bead-purified CD3+ T cells in the presence of a neutralizing antibody against human PD-L1 (2 μg/ml) (R&D Systems).
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6

Quantifying Cell Internalization by Flow Cytometry

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To quantitatively assess cell internalization, Sen or Res cells were labelled with 3 μM CFSE (carboxyfluorescein diacetate succinimidyl ester) dye (Stemcell Technologies, VIC, Australia) for 10 min at 370 C in serum-free RPMI 1640. Labelling was stopped with complete media and the cells were washed twice prior to co-culture with macrophages. 5×104 THP-1 macrophages were co-cultured (ratio of 1:1) with the CFSE labelled Sen or Res or D3 cells. 50 μg of Res-MP or Sen-MP or D3-MPs were added to the heterotypic cell cultures and following 24 h incubation cells were harvested and stained with a macrophage marker, APC conjugated anti-CD11b antibody (BD Biosciences). Samples were incubated for 30 min in the dark, washed twice in PBS and analysed for dual labels and single labels with the BD LSR Fortessa™ X-20 flow cytometer. The cells which were dual positive for both markers (CFSE-green channel and CD11b-red channel) represent those cells engulfed by macrophages. The remainder of the population comprises of macrophages alone, cells which have engulfed macrophages or cells alone. This was measured by the percentage drop in the population of each of these in their respective channels.
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7

CFSE-based Lymphocyte Proliferation Assay

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The carboxy uorescein diacetate succinimidyl ester (CFSE, Biolegend, USA) was used to label the proliferation of the PBMCs. The cells were resuspended with 5 µM CFSE working uid and incubated in a carbon dioxide cell incubator for 20 min. Next, a ve-time volume of the RPIM1640 medium containing 10% fetal bovine serum was added to terminate the reaction, and then washed three times by RPIM1640 medium. After labeling with CFSE, the cells were incubated with human CD3/CD28 T cell activator (Stem Cell, Canada) at a dose of 25 µL/mL and IL-2 (Pepro Tech, USA) at a dose of 50 ng/mL for up to 3 d, and then analyzed by uorescence-activated cell sorting (FACS).
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