cell type: oral keratinocyte time point: control tissue: tongue strain: BALB/c
Treatment protocol
BALB/c mice were sacrificed and the tongues, inner part of the cheeks and plates were obtained. After trimming submucosal tissue attached to the oral mucosa, the tongues, plates and cheeks were immersed in dispase II (1.2 units/mL from Roche, Lewes,UK) to be digested at 4 °C overnight. Subsequently, the epithelial layer was enzymatically and mechanically separated from the underlying connective tissue and was further incubated in 0.25% Trypsin/0.02% EDTA (Gibco, Grand Island, NY, USA) for 3-5 min with vigorous pipetting to achieve cell dispersion. After trypsin was inactivated by 10% fetal bovine and washing, the cell suspension was re-suspended in PBS to the concentration of 1×107 cells/ml. After being intraperitoneally anesthesized with chloral hydrate, C57BL/6J mice were given a subcutaneous injection with 50 μl cell suspension obtained from BALB/c mice into the foreneck. Seven days later, 25μl cell suspension was injected to the same C57BL/6J mouse into tongue submucosa under anesthesia. Groups of six C57BL/6J mice at 48h and 96h after tongue submucosal injection and control without any manipulation were sacrificed. Tongues were excised and subcutaneous tissue and blood were trimmed. The epithelia were obtained after being digested in dispase II (1.2 U/mL from Roche, Lewes,UK) at 4 °C overnight. Subsequently, the epithelial layer was enzymatically and mechanically separated from the underlying connective tissue. The epithelial layer was further incubated in 0.25% Trypsin/0.02% EDTA (Gibco, Grand Island, NY, USA) at 37 °C for 3-5min with vigorous pipetting to achieve cell dispersion. After trypsin neutralization by fetal calf serum (FCS) and washing, the keratinocytes were collected for the following experiments.
Growth protocol
tongue mucosa keratinocyte from 6-8 weeks old C57 mouse
Extracted molecule
total RNA
Extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA
Hybridization protocol
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse 430 2.0 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. scan protocol GeneChips were scanned
Scan protocol
using the GeneChip Scanner 3000 7G
Description
gene expression data from oral keratinocyte during adaptive immune response
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. The trimmed mean target intensity of each array was arbitrarily set to 500.
