TALL-104 human leukemic CTLs were maintained as described previously 9 (link). Early experiments were conducted in complete culture medium. Later experiments were in culture medium without IL-2. Complete culture medium contains 40mL heat-activated fetal bovine serum, 4mL Pen-Strep, 8mL L-Glutamine, 80 μL IL-2, and 348mL Iscove’s modified Dulbecco’s medium. Dynabeads polystyrene beads coated with anti-CD3 or anti-CD8 monoclonal antibody (Thermo Fisher, Waltham, MA) were washed in medium according to the manufacturer’s protocol, and were added to the cell suspension at a ratio of ~one bead per cell. Alexa647-conjugated anti-LAMP1 antibody (0.5 μg/mL final concentration) was purchased from BD Pharmingen (San Jose, CA). Phorbol 12-myristate 13-acetate (PMA) was from Alexis Biochemicals (San Diego, CA). Thapsigargin (TG) was from AdipoGen (San Diego, CA). Cells were incubated with imiquimod (Adipogen) San Diego, California) at 10 uM or PMA at 100 nM for indicated time. The National Cancer Institute’s Diversity Set V (https://wiki.nci.nih.gov/display/NCIDTPdata/Compound+Sets ) was used for screening, and powder resupply was from NCI. Compounds were prepared in DMSO at a final concentration of 10μM.
Phorbol 12 myristate 13 acetate pma
Manufactured by Alexis Biochemicals
Phorbol 12-myristate 13-acetate (PMA) is a chemical compound commonly used as a laboratory reagent. It serves as a potent activator of protein kinase C (PKC), a family of enzymes involved in various cellular signaling pathways. PMA is a well-established tool for researchers to investigate the role of PKC in a wide range of biological processes.
Automatically generated - may contain errors
2 protocols using phorbol 12 myristate 13 acetate pma
1
Leukemic CTLs Maintenance and Assays
2
Fluorescent Biosensors in Hematopoietic Cells
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TALL-104 cells were obtained from the ATCC and cultured and transfected using nucleofection as described previously. 18 K562 cells were obtained from the ATCC and cultured in Iscove's modified Eagle's medium (MEM) supplemented with 10% heat-inactivated serum, glutamine, and penicillinstreptomycin, and were transfected using nucleofection according to the manufacturer's recommended protocol. CDNAs encoding EKAR, 19 CKAR, 2 DAGR, 2 DKAR, 20 and D3CPV 21 were obtained from Addgene (see Suppl. Table for a description of each sensor). The JNKAR construct 22 was provided by Dr. Jin Zhang. To generate polyclonal long-term transfectants, K562 cells were transiently transfected, then sorted on YPF fluorescence ~2 weeks and ~4 weeks after transfection. Experiments with transiently transfected TALL-104 cells were conducted in normal Ringer's solution. 18 All other experiments were conducted in complete medium. Phorbol 12-myristate 13-acetate (PMA) was from Alexis Biochemicals (San Diego, CA). Thapsigargin (TG) was from AdipoGen (San Diego, CA). Anisomycin was from Sigma Chemical (St. Louis, MO). Calcein red-orange and Far Red DDAO-SE were from Molecular Probes (Eugene, OR).
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