|
| Status |
Public on Mar 08, 2011 |
| Title |
ENK_Donor_14 |
| Sample type |
RNA |
| |
|
| Source name |
enriched expanded natural killer cells from healthy donor
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Donor 14
cell type: natural killer cells
nkc state: expanded
|
| Growth protocol |
Expansion Protocol: Peripheral Blood was centrifuged over Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ). PBMCs were harvested and washed in RPMI1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Invitrogen). PBMCs from the donor and myeloma patient were co-cultured with irradiated (100 gray) K562-mb15-41BBL cells at a ratio of 1.5:1 in RPMI 1640 + 10% FBS + 300 IU/ml of IL-2 (Prometheus, San Diego, CA). One half the media was changed every other day with fresh media containing IL2. The cells were collected on day 10. The number of viable cells was based on counts using trypan blue exclusion.
NK Enrichment Protocol: NK cells were isolated from fresh PBMCs from healthy donors using customized NK cell enrichment kit (catalog # 19055) from Stem Cell Technologies Inc. (Vancouver, BC). In brief, 10x107 cells were incubated with 200μl enrichment cocktail for 10-15 minutes at room temperature. Magnetic microparticles/nanoparticles were added to the cells and incubated for 10 minutes. Cell suspension was then placed into the EasySep® Magnet for 5 minutes. The magnetically labeled unwanted cells remained bound inside the original tube. Unlabeled NK cell fraction was collected into a new tube. Purity and viability of enriched NK fraction was assessed by trypan blue exclusion and flow cytometry analysis for NK cell surface markers. A similar process was performed for the enrichment of ENK cells from expanded cells. For NK/ENK enrichment from myeloma patients, 10μl CD138TAC was added to the enrichment cocktail to remove any myeloma cell contaminants.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from ~95% purified NK/ENK cells (2-5M cells) using the Qiagen RNeasy® Mini Kit (Qiagen, Catalog # 74104) as per instructions. The samples were eluted in RNase free water and quantitated using a NanoDrop 1000 Spectrophotometer (Thermo Scientific Inc. Wilmington, DE).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the New GeneChip 3 IVT Labeling Kit protocol from 500 ng of total RNA (The New GeneChip® IVT Labeling Kit: Optimized Protocol for Improved Results, 2004, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 µg of cRNA were hybridized for 16 hr at 45ºC on HG-U133plus 2.0 gene chip microarrays (Affymetrix, Santa Clara, CA).
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000. Scanned images were processed using the GCOS v1.4 software.
|
| Description |
HD_ENK_14
Gene expression data from donor expanded NK cells
|
| Data processing |
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method.
|
| |
|
| Submission date |
Mar 08, 2011 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Tarun Kumar Garg |
| E-mail(s) |
gargtarunk@uams.edu
|
| Phone |
501-526-6990-8482
|
| Organization name |
University of Arkansas for Medical Sciences
|
| Department |
Myeloma Institute for Research and Therapy
|
| Lab |
Immunotherapy Lab
|
| Street address |
4301 W Markham St
|
| City |
Little Rock |
| State/province |
AR |
| ZIP/Postal code |
72205 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE27838 |
Gene expression of expanded and non-expanded natural killer cells from healthy donor and myeloma patients |
|
| Relations |
| Reanalyzed by |
GSE86362 |
