PS1 FBS Min
Homo sapiens
Protocols: The human subcutaneous adipose tissue was sampled from 5 plastic surgery patients (PS1, PS6, PS7, PS8, PS9). The procedure was approved by the Independent Bioethics Commission for Research of the Medical University of Gdansk (NKBBN/387/2014) and written informed consent was obtained from the patients prior to surgery. The isolation of adipose-derived stem cells (ASCs) was based on standard protocol. Briefly, following enzymatic digestion and erythrocyte lysis, the cells were suspended in culture medium Dulbecco's modified Eagle medium (DMEM low glucose - 1000 mg) supplemented with 10% fetal bovine serum FBS (Sigma-Aldrich, Saint Louis, Missouri, USA). After 24h in culture condition (37ºC, 5% CO2) non-adherent and non ASCs cells were removed). ASCs were seeded on special Primaria flasks (Corning, USA) in amount of 375 000 cells/75cm2 flask. The primary cell culture was named culture P0 (cells before passage). In order to assess the influence of serum-deprivation on the transcriptomic profile of ASCs cells after the 2nd passage (P2) were seeded and cultured for 24h in media supplemented with 10% FBS (adherence). After 24h the media were changed and cells were cultured in two different conditions: 1) “serum-deprivation condition” - 24h incubation with 5% of FBS (cell adaptation) followed by FBS deprivation (0% FBS) for next 48h; 2) “standard condition” - 72h incubation with 10% FBS. After this time cells were trypsynised, collected, centrifuged and the pellet was snap frozen for NGS analysis. ASCs (3-5x 105 of cells) after passage 2(P2) were trypsynised, washed, and snap frozen for transcriptomic analysis. The pellets were stored in - 70ºC prior to RNA isolation. RNA was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to original protocol with two modifications: (i) 1-bromo-3-chloropropane was used instead of chloroform to prevent foaming and emulsification and (ii) the elution was carried out with 40µl of water followed by repeated elution with the entire volume of the original eluate. Quality (RNA Integrity Number - RIN) and concentration of samples were assessed using Bioanalyzer 2100 Instrument and RNA 6000 Nano Kit (Agilent, Waldbronn, Germany). Samples with RINs value >8.0 were used for further transcriptomic analysis. Ribosome reduction kit (Arraystar Inc, Rockville, USA) and SureSelect Strand Specific mRNA library kit (Agilent, Santa Clara, USA) were used according to standard procedure. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Library Quantification Kit for Illumina (Kapa Biosystems, Woburn, USA).
ENA-FIRST-PUBLIC
2018-08-09T17:03:22Z
ENA-LAST-UPDATE
2018-08-09T11:28:36Z
External Id
SAMEA4823072
INSDC center name
Medical University of Gdansk Department of Biology and Pharmaceutical Botany
INSDC first public
2018-08-09T17:03:22Z
INSDC last update
2018-08-09T11:28:36Z
INSDC status
public
Submitter Id
E-MTAB-6436:PS1 FBS Min
age
53
broker name
ArrayExpress
cell type
adipose tissue derived mesenchymal stem cell
common name
human
disease
normal
growth condition
FBS-deprivation condition
individual
PS1
organism part
subcutaneous adipose tissue
sample name
E-MTAB-6436:PS1 FBS Min
scientific_name
Homo sapiens
sex
female