Ex vivo-purified primary CD4 and CD8 T cells were obtained from the blood of a healthy donor using Rosettesep CD4 and CD8 T cell enrichment cocktails, respectively (Stemcell Technologies). Primary T lymphocytes were maintained in RPMI 1640 supplemented with 10% FBS and 30 U of rIL-2/ml. T cells were expanded using Dynabeads human T-activator CD3/CD28 (Life Technologies) immediately after isolation. The purity and activation status of CD4 and CD8 T cells were determined by using flow cytometry and antibodies against CD4 (BD Biosciences), CD8 (BioLegend, clone RPA-T8), CD3 (BD Pharmingen), and CD25 (BioLegend, clone BC96). The resting cell population was removed from T-cell receptor (TCR) stimulation 24 h prior to the experiment, and the activation status was determined at the time of plating for the viability test. Viability was determined using the fixable viability dye eFlour 660 (eBioscience) at 24 hpi with VSV-XN2 or VSV-gp160G at the indicated MOI. Activated primary CD4 T cells were also infected with wild-type VSV Indiana serotype at an MOI of 0.01 at 24 hpi and measured for viability compared to VSV-XN2 and VSV-gp160G. Prior to analysis, cells were fixed in 2% paraformaldehyde. Cells were analyzed using LSR-Fortessa-HTS.
Eflour 660
Manufactured by Thermo Fisher Scientific
The EFlour 660 is a compact, high-performance analytical instrument designed for the sensitive detection and quantification of fluorescent molecules. It utilizes a solid-state laser excitation source and a high-sensitivity photon detection system to provide reliable and reproducible fluorescence measurements. The EFlour 660 is suitable for a variety of applications in life science research and clinical diagnostics, where accurate and sensitive fluorescence detection is required.
Automatically generated - may contain errors
2 protocols using eflour 660
1
T cell viability upon VSV infection
2
Efficient iPSC-derived NK Cell Expansion
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Briefly, a two‐step protocol was adopted via EB formation. According to the instructions, iPSCs were seeded onto the Aggrewell 800 (STEMCELL Technologies #34850). After a 10‐day half‐medium change with EB formation medium, EBs were transferred to gelatin‐coated dishes, and half‐medium changes were made twice a week after the first week. To remove any clumps, cells can be collected via a 40 µm cell strainer (Corning #431750).
Biotinylated IL21 (Acro Biosystems #IL1‐H82F7) and biotinylated 4‐1BBL (Acro Biosystems #41L‐H82F9) bound streptavidin magnetic beads (Invitrogen #91216845) were employed to stimulate the expansion of NK cells. Beads were added to NK cells at a 1:1 ratio. Medium was replaced every 2‒3 days with NK cell expansion medium freshly added with 500 IU/mL IL2 and 10 ng/mL IL15. The coupling results of beads were tested using flow cytometry: IL21 (eFlour660; eBioscience) and 4‐1BBL (PE‐CY7; eBioscience).
To characterize the iPSC‐derived NK cells, flow cytometry was used to evaluate the expression level of the NK cell surface markers. The cell culture medium and antibodies are listed in the Supporting Information.
Biotinylated IL21 (Acro Biosystems #IL1‐H82F7) and biotinylated 4‐1BBL (Acro Biosystems #41L‐H82F9) bound streptavidin magnetic beads (Invitrogen #91216845) were employed to stimulate the expansion of NK cells. Beads were added to NK cells at a 1:1 ratio. Medium was replaced every 2‒3 days with NK cell expansion medium freshly added with 500 IU/mL IL2 and 10 ng/mL IL15. The coupling results of beads were tested using flow cytometry: IL21 (eFlour660; eBioscience) and 4‐1BBL (PE‐CY7; eBioscience).
To characterize the iPSC‐derived NK cells, flow cytometry was used to evaluate the expression level of the NK cell surface markers. The cell culture medium and antibodies are listed in the Supporting Information.
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