|
| Status |
Public on Jan 23, 2024 |
| Title |
CAR_3 |
| Sample type |
protein |
| |
|
| Source name |
Microglia isolated from CD19 CAR group
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Microglia
tissue: Brain
|
| Treatment protocol |
100000 B-NHL (A20) cells were injected iv to sublethally irradiated mice. On day 7, 10 Million CD19 CAR T cells were injected iv and mice were sacrificed on day 10. Brain was isolated and FACS sorted for microglia (CD45lo and CD11b) cells.
|
| Growth protocol |
B-NHL cells were maintained in DMEM complete media with 10% FCS and Penstrip
|
| Extracted molecule |
protein |
| Extraction protocol |
PamGene lysates were prepared according to manufacturers instructions with M-PER Mammalian Protein Extraction Reagent (ThermoFisher) containing Halt phosphatase and Halt protease inhibitor cocktails (ThermoFisher). Total protein was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher).
|
| Label |
PY20 FITC
|
| Label protocol |
We used the STK PamGene Array according to instructions
|
| |
|
| Hybridization protocol |
5 μg of protein per well was loaded onto the PamChip and the assay was run on a PamStation96 (PamGene International).
|
| Scan protocol |
The degree of phosphorylation was measured in real time via the kinetic image capture program, Evolve (PamGene International) Following 60 min incubation, the signal intensities for each peptide were analyzed using BioNavigator Software (PamGene International).
|
| Description |
Protein lysates with still active kinases, phosphorylation of spotted peptides on chip detected with FITC labeled anti-phospho antibody
|
| Data processing |
Only peptides that showed kinetics (increase of signal in time) on at least 25 % of the arrays were included in the analysis. Nominal coefficient of variation (CV) was calculated per peptide using a 2-component error fit model using overall mean as input and was used as a filter to remove low-intensity spots. Only peptides that showed nominal CV <0.5 were included in the analysis. For kinase interpretation, a ranked list of putative kinases responsible for differences in the peptide phosphorylation was generated by upstream kinase analysis (UKA, PamGene International).
|
| |
|
| Submission date |
Feb 22, 2023 |
| Last update date |
Jan 23, 2024 |
| Contact name |
Robert Zeiser |
| Organization name |
Uniklinik_Freiburg
|
| Department |
Inner Medicine 1
|
| Street address |
Hugstetter Strasse 53
|
| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL33157 |
| Series (1) |
| GSE225811 |
Signaling in microglia during CAR Tcell induced Immune cell associated neurotoxicity syndrome (ICANS) |
|
