|
| Status |
Public on Aug 03, 2022 |
| Title |
CD4-Treated TAMs - technical repeat 1 - mAdbID:116689 |
| Sample type |
RNA |
| |
|
| Source name |
CD4-treated TAMs
|
| Organism |
Mus musculus |
| Characteristics |
strain backgroud: C57BL/10
genotype/variation: rag-/- (Rag KO)
cell type: Tumor Associated Macrophages (TAMs)
|
| Treatment protocol |
In-vivo treatment: Tumor bearing RAGKO mice were first challenged with 10(5) MB49 tumor cells s.c.. Five days later, half of the mice received Marilyn CD4 T cells i.p. All mice were euthanized 12 days after tumor challenge. The tumors were dissociated and cells stained with different antibodies to either compare the phenotype of Untreated versus CD4-treated TAMs or to compare their protein production. For the latter, between 10(5) and 1.25x10(5) TAMs sorted TAMs were plated in 200 uL of complete IMDM in 96 well-plates. Supernatants were collected 18 hours later and sent to Raybiotech, Inc. to measure cytokines and chemokines characteristic of M2 or M1 macrophages using a custom Cytokine array. Amount of protein in the supernatant was normalized to 10(5) cells. For the microarray experiments, sorted TAMs were lysed immediately post sort for RNA isolation. TAMs were sorted based on the markers CD11b+, MHC ClassII+, Gr-1neg and 7AADneg.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen miRNeasy Mini Kit Protocol
Sorted TAMs were lysed immediately post sort for RNA isolation using miRNeasy mini kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
NuGEN Biotinylation Protocol
RNA quality was verified by Bioanalyzer with RNA Integrity Number greater than 8.5 for all samples. Microarray target material was prepared by amplification of 50 ng total RNA using NuGEN WT-Ovation systems V2, followed by biotinylation with NuGEN Encore(TM) BiotinIL, using 4ug input of cDNA.
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| |
|
| Hybridization protocol |
Illumina Hybridization Protocol
We applied 2 ug of conjugate to Illumina Mouse WG-6 v2.0 Expression BeadChip microarrays and hybridized at 48 degree Celsius for 16 hr, post-processed per manufacturer's instructions.
|
| Scan protocol |
Illumina Scanning Protocol
Illumina Mouse WG-6 v2.0 Expression BeadChip microarrays were scanned using a HiScan (Illumina). Images were analyzed using Genome Studio software (Illumina).
|
| Description |
mAdb experiment ID: 116689
|
| Data processing |
BRB ArrayTool Data Processing
Calculation Method: Microarray data were analyzed using BRB Array Tools developed by the Biometric Research Branch of the National Cancer Institute under the direction of Dr. Richard Simon (http://linus.nci.nih.gov/BRB-ArrayTools.html). Array data were filtered to threshold the spot intensity of the probes at the minimum value if the spot intensity was <10, and a quantile normalization was applied.
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| |
|
| Submission date |
Sep 17, 2012 |
| Last update date |
Aug 03, 2022 |
| Contact name |
Ainhoa Perez-Diez |
| E-mail(s) |
ainhoa@nih.gov
|
| Phone |
(301) 496-3503
|
| Organization name |
NIAID, NIH
|
| Lab |
Lab of Immunoregulation
|
| Street address |
9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL6887 |
| Series (1) |
| GSE40920 |
CD4 cells re-educate tumor associated macrophages. |
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