|
| Status |
Public on Jul 17, 2023 |
| Title |
NK cells cultured alone, rep 4 |
| Sample type |
RNA |
| |
|
| Source name |
Mus musculus liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
age: 12 weeks
tissue: liver
cell type: NK cells
treatment: cultured alone
|
| Treatment protocol |
NK cells isolated from the liver using flow cytometric sort (gated as live CD45+CD3negNK1.1+NKp46+CD200RnegTRAILneg cells) were cultured alone or co-cultured with purified LSECs for 14-16 hours, and then sorted (> 99% of live CD45+CD3negNK1.1+NKp46+ cells) for gene expression analysis.
|
| Growth protocol |
Livers were perfused with PBS, and mechanically and enzymatically digested using Liver Dissociation Kit and gentleMACS™ Octo Dissociator (Miltenyi). LSECs were purified from dissociated liver tissue by density gradient centrifugation, followed by magnetic separation with anti-CD146 microbeads (Miltenyi). Purified LSECs were cultured on collagen-coated plates (Gibco) in DMEM (Sigma) supplemented with 10% FCS, 1% Penicillin/Streptomycin, 1% L-glutamine, 1% MEM non-essential amino acids, 1 mM sodium pyruvate and 50 µM β-mercaptoethanol (all from Gibco). 24 h after plating, media was removed, and NK cells were added.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using RNeasy Mini Kit (Qiagen), and genomic DNA was removed by TURBOTM-DNase (Thermo Fisher Scientific)
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
|
| |
|
| Hybridization protocol |
Hybridization (16h x 45°C) was processed according to the standard Affymetrix protocol.
|
| Scan protocol |
Affymetrix GeneArray Scanner3000
|
| Description |
NK_4
|
| Data processing |
The data were analyzed with a commercial software called JMP Genomics, version 15, from SAS. Gene expression profiling was performed using arrays of Mouse Clariom D from Thermo Fischer Scientific. A Custom CDF Version 22 with Entrez based gene definitions was used to annotate the arrays. The Raw fluorescence intensity values were normalized applying quantile normalization, RMA background correction and Medianpolish Probeset Summary
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| |
|
| Submission date |
May 23, 2023 |
| Last update date |
Jul 17, 2023 |
| Contact name |
Carolina Delatorre |
| E-mail(s) |
carolina.delatorre@medma.uni-heidelberg.de
|
| Organization name |
University Heidelberg
|
| Street address |
Theodor-Kutzer-Ufer 1-3
|
| City |
Mannheim |
| ZIP/Postal code |
68167 |
| Country |
Germany |
| |
|
| Platform ID |
GPL32874 |
| Series (1) |
| GSE233222 |
Gene Expression Analysis of NK cells after co-culture with Liver Sinusoidal Endothelial Cells (LSECs) |
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