|
| Status |
Public on Aug 03, 2018 |
| Title |
SK04_JK CD63+ |
| Sample type |
RNA |
| |
|
| Source name |
Exosome bound by CD63
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Jurkat cell line exosomes
tissue: Myeloid
|
| Treatment protocol |
The cultured Jurkat were washed with IXPBs followed by a second wash with HITES medium (DMEM/F12 supplemented with, bovine serum albumin, hydrocortisone, insulin, transferrin, and trace elements). EVs were isolated from Jurkat T cells were cultured overnight using HITES medium. The conditioned media from Jurkat T cells were collected, and cell debris were removed using centrifugation at 300 g for 5 minutes and 2500 g for 10 minutes respectively. Centrifuged media were concentrated using Ultra-4 centrifugal filters to about 1 ml volume and further centrifuged at 10,000 rpm for 10 minutes. EVs were isolated using an Exo-Quick kit (SBI) by incubating overnight at 4°C. CD63 and CD47 positive EVs were captured using Biotin conjugated antibodies.
|
| Growth protocol |
The Jurkat T cell line (E6.1) was purchased from ATCC. The cells were maintained using RPMI 1640 containing 10% FBS, glutamine, penicillin, and streptomycin (Gibco). The Jurkat and JinB8 T cells used for experiments were maintained less than 6 weeks in culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (Invitrogen) and RNaseazy (Qiagen) kits were used to extract total RNA according to the manufacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
Labeling was performed on a sample-by-sample basis according to manufacturer’s guidelines for use with the Affymetrix microRNA GeneChips version 4.0 (Affymetrix, Inc). Bioanalyzer nanochip (Agilent, Inc) and NanoDrop (ThermoFisher, Inc) were used to validate and quantitate the RNA prior to labeling. 300ng of total RNA was used for labeling with the Affymetrix FlashTag kit (cat#901910).
|
| |
|
| Hybridization protocol |
After hybridization, the probe arrays were stained with streptavidin-phycoerythrin solution (Molecular Probes) and enhanced using an antibody solution containing 0.5 mg/mL of biotinylated anti-streptavidin (Vector Laboratories).
|
| Scan protocol |
Genechips were scanned using an Affymetrix Gene Chip Scanner 3000.
|
| Data processing |
Gene expression intensities were calculated using GeneChip Command Console Software (AGCC). .cel files generated by the Affymetrix AGCC program were imported in the Affymetrix Expression Console software and RMA (Robust Multichip Analysis) normalization was performed to generate the .chp files. The .chp files were normalized, log2 transformed, and summarized.
|
| |
|
| Submission date |
Aug 30, 2017 |
| Last update date |
Aug 03, 2018 |
| Contact name |
abdel G Elkahloun |
| E-mail(s) |
abdel@mail.nih.gov
|
| Phone |
301 402 3170
|
| Organization name |
NHGRI-NIH
|
| Lab |
MICROARRAY CORE
|
| Street address |
50, SOUTH DRIVE
|
| City |
BETHESDA |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL21572 |
| Series (1) |
| GSE103310 |
Jurkat cell exosomes bound by either CD47 or CD63 harbor different RNA content |
|
