A subclone of HEK 293 cells, called HEK 293a, originally obtained from Sigma-Aldrich was used in these experiments, either as a non-transfected cell type or as one stably expressing myc-tagged human B2Rs [8 (link)]. These cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 1 % l -glutamine, and 1 % penicillin–streptomycin stock solutions (100×). Cells were detached using the protease-free Cell Dissociation Buffer (Invitrogen), incubated in D-MEM without serum at 37 °C for 30 min under agitation in the presence of a Cy7-conjugated ligands and other drugs, rapidly centrifuged (30 s, 11,000g) and resuspended in phosphate buffered saline. Then, the fluorescence of the cell suspensions was assessed using the BD SORP LSR II cell analyzer for the uptake of the as a function of stimulation and transgene expression. The cytofluorometry results were analyzed using the BD FACS DIVA software.
Hek 293a
Manufactured by Merck Group
The HEK 293a is a laboratory cell line derived from human embryonic kidney cells. It is commonly used in cell culture research and experimentation due to its reliable growth characteristics and ease of transfection. The HEK 293a cell line provides a standardized model for a variety of in vitro studies.
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Lab products found in correlation
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Hrp conjugated secondary antibodies against rabbit igg, by Cell Signaling Technology (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
Hek293a cells, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
2 protocols using hek 293a
1
Fluorescence-based Ligand Uptake Assay
2
CRISPR-Cas9 Mediated LRRC8A Knockout in HEK293A Cells
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HEK293A cells (Invitrogen) and LRRC8A-knockout HEK293A cells were cultured in DMEM-high glucose (Sigma-Aldrich, Cat. #D5671), supplemented with 10% fetal bovine serum (FBS; BioWest, Cat. #S1560-500) and 100 units/ml penicillin G (Meiji Seika, Cat. #6111400D2039). All cells were cultured in a 5% CO 2 atmosphere at 37ºC and verified to be negative for mycoplasma. LRRC8A-knockout HEK293A cells were established using the CRISPR-Cas9 system 39 . Briefly, the targeting sequence for human LRRC8A, 5′-GCACAACATCAAGTTCGACGTGG-3′, was designed using the CRISPR design tool (http://crispr.mit.edu/), and oligo DNAs for its sense and anti-sense were purchased from Bethyl Laboratories, MBL, and Sigma-Aldrich, respectively.
HRP-conjugated secondary antibodies against rabbit IgG (Cat. #7074, 1:2,000) and mouse IgG (Cat. #7076, 1:10,000-1:20,000) were purchased from Cell Signaling Technology.
HRP-conjugated secondary antibodies against rabbit IgG (Cat. #7074, 1:2,000) and mouse IgG (Cat. #7076, 1:10,000-1:20,000) were purchased from Cell Signaling Technology.
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