LCMV-immune mice with memory P14 Tg CD8+ T cells were generated as described above and subsequently implanted with EO771.ova cancer cells. After 14–20 days of tumor growth, tumors and spleen were processed as described. Cells were then cultured in a 96-well flat bottom plate (120338, Globe Scientific) at a concentration of 10 × 106 cells/mL in a base RPMI media with 10% FBS, 100U/mL penicillin/streptomycin, 1x MEM non-essential amino acids (11140050, Gibco), 1mM HEPES, 40mM L-glutamine, 1:1000 2-Mercaptoethanol (21985023, Gibco), and 1mM sodium pyruvate (11360070, Gibco). Each sample was cultured for 4 hours at 37°C under the following four conditions: 1) base media alone, 2) 2μM GP33–41 peptide from LCMV (KAVYNFATM), 3) 5μM OVA257–264 peptide from ovalbumin (SIINFEKL), or 4) 1:500 phorbol 12-myristate 13-acetate (PMA) and ionomycin cocktail (00–4970-93, eBioscience).
Ionomycin cocktail
Manufactured by Thermo Fisher Scientific
Ionomycin cocktail is a laboratory reagent used to induce cellular signaling and activation in various cell types. It acts as a calcium ionophore, facilitating the influx of calcium ions into the cell. The core function of Ionomycin cocktail is to serve as a tool for researchers to study calcium-dependent cellular processes.
Automatically generated - may contain errors
2 protocols using ionomycin cocktail
1
Comparing LCMV-specific CD8+ T cell Responses
2
Profiling Immune Gene Expression in SLE
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Gene expression profiling was performed by NanoString, using the pre-designed nCounter Human Immunology v2 Panel (NanoString Technologies). Total CD4+ T cells from a subset of (i) four SLE patients with previously identified high transcriptional IFN signature, (ii) two SLE patients with low IFN signature, and (iii) four healthy controls, were obtained by negative selection (StemCell Technologies) from cryopreserved PBMCs. A total of 25,000 cells were collected into RLT lysis buffer (Qiagen) following in vitro stimulation for 120 min with PMA and ionomycin cocktail without addition of protein transport inhibitors (eBiosciences). RNA was extracted using the RNAeasy Micro Plus kit (Qiagen), with gDNA cleanup, and hybridized to the NanoString CodeSets, following manufacturer's instructions. Expression levels were assessed using an nCounter Sprint instrument (NanoString Technologies). Data were processed using the nSolver Analysis Software following normalization of the raw read counts to the geometric mean of positive control spike-ins, and the gene expression of 15 selected housekeeping genes that were found to have low variability following in vitro stimulation, as previously described (28 (link)).
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