104 microgels, with or without biotin, were co-incubated for 30 min in 500 µL PBS with 1% bovine serum albumin containing varying concentrations of SA-FasL. Microgels were washed 8 times by centrifugation to remove unbound SA-FasL, and were incubated with 106 A20 cells (ATCC) in 1.0 mL media. After 18 h, cells were stained with markers of early and late apoptosis (annexin V-APC and propidium iodide, BD Biosciences). Samples were analyzed by flow cytometry (Accuri C6 flow cytometer) and cells staining positive for either marker were considered apoptotic. Three independent runs of this experiment were performed with consistent results.
A20 cells
Manufactured by American Type Culture Collection
The A20 cells are a mouse B cell line derived from a spontaneous lymphoma. They are commonly used as a model system for studying B cell signaling and biology.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by BD (2 mentions)
Annexin 5 apc, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Phoenix eco packaging cells, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Balb cannhsd mice, by Inotiv (1 mentions)
4 protocols using a20 cells
1
Apoptosis Induction in A20 Cells
2
Cell Line Maintenance: A20 and Phoenix-ECO
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A20 cells (catalog number TIB-208) and Phoenix-ECO packaging cells (catalog number CRL-3214) were purchased from ATCC. All cell lines and culture experiments were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS, nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin, 11 mM glucose, and 2 μM 2-mercaptoethanol. Cell lines were routinely tested for potential mycoplasma contamination.
3
Apoptosis Induction in A20 Cells
4
In Vivo Tumor Microinjection Profiling
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A20 cells (ATCC) were cultured in RPMI 1640 with l -Glutamine (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), and 50 nM β-Mercaptoethanol at 37 °C and 5% CO2. All experiments in mice were approved by IACUC Board of Presage Biosciences, Seattle, WA (Protocol number PR-001) and were performed at Presage in accordance with relevant guidelines and regulations. For generating A20 allografts, female BALB/cAnNHsd mice (Envigo) were inoculated with 1 × 106 A20 cells. CIVO IT microinjections were performed as described previously28 (link). Briefly, mice (n = 6 per time point, 4 and 24 h) were enrolled in microinjection studies when implanted tumors reached the following approximate dimensions: 14 mm (length), 10 mm (width) and 7 mm (depth). The CIVO device was configured with 6 thirty-gauge injection needles with a total volume delivery of 2 μL. Presage’s fluorescent tracking marker (FTM, 5% by volume) was added to the injection contents for spatial orientation. At 4 and 24 h following CIVO microinjections, mice were euthanized using CO2 inhalation for biomarker analyses.
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