CP5
Homo sapiens
Protocols: Peripheral blood leukocyte cones from healthy human donors were acquired from the National blood service under ethics license REC 05/Q0401/108 (University of Manchester). Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation using Ficoll-Paque (GE Healthcare; Life Sciences) and NK cells isolated by negative selection (NK cell isolation kit; Miltenyi Biotech). NK cells were cultured at 37⁰C and 5% CO₂ in Dulbecco's Modified Eagle Medium (DMEM) medium (Sigma) containing 30% Ham's F-12, 10% human serum (Sigma), 1% nonessential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine, 50 U/mL penicillin-streptomycin, and 50 μM 2-mercaptoethanol (Gibco) (NK cell clone medium). NK cells were also supplemented with 200 U/mL interleukin-2 (Roche) and rested for 6 days prior to experiments. To investigate the effects of PGE₂, NK cell cultures were additionally supplemented with 500 ng/mL synthetic PGE₂ (Sigma) 24 hours prior to cell harvesting and lysis. NK cells were cultured at 37⁰C and 5% CO₂ in Dulbecco's Modified Eagle Medium (DMEM) medium (Sigma) containing 30% Ham's F-12, 10% human serum (Sigma), 1% nonessential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine, 50 U/mL penicillin-streptomycin, and 50 μM 2-mercaptoethanol (Gibco) (NK cell clone medium). NK cells were also supplemented with 200 U/mL interleukin-2 (Roche) and rested for 6 days prior to experiments. After being rested for six days the NK cells were left untreated or supplemented with 500 ng/mL synthetic PGE₂ (Sigma) 24 hours prior to cell harvesting and lysis. NK cells were re-suspended in RLT buffer (RNeasy lysis buffer; Qiagen) containing 0.01% β-mercaptoethanol to lyse cells and stored at -80⁰C prior to RNA extraction. RNA was extracted from thawed samples using the RNeasy mini kit (Qiagen) as per the manufacturer's instructions. mRNA libraries were prepared using the Illumina stranded mRNA prep kit.
ENA-FIRST-PUBLIC
2023-11-28T17:00:04Z
ENA-LAST-UPDATE
2023-11-28T17:00:04Z
External Id
SAMEA113907502
INSDC center name
UOM
INSDC first public
2023-11-28T17:00:04Z
INSDC last update
2023-11-28T17:00:04Z
INSDC status
public
Submitter Id
E-MTAB-13106:CP5
broker name
ArrayExpress
cell_type
natural killer cell
collection date
not collected
common name
human
developmental stage
adult
disease
normal
geographic location (country and/or sea)
not collected
individual
3
organism part
blood
sample name
E-MTAB-13106:CP5
scientific_name
Homo sapiens