Application
Flow Cytometry
Sample
Human Cell (Cell lines: HL60 and NB4)
Permeabilization
Yes - 90% methanol (diluted in PBS)
Gating Strategy
Intact cells (Event Length vs 191Ir (DNA channel) --> 191Ir vs 193Ir (DNA channel). Caspase-3 cleaved positive cells were removed by gating.
Specification
Cell lines: HL60 and NB4
Preparation
Cell harvesting/tissue preparation method: Purchased suspension cell line. In culture at 37C, 5% CO2.
Sample buffer: In culture with RPMI medium supplemented with 10% FBS, 1% Penicillin-Streptomycin and 1%L-Glutamine.
Sample buffer: In culture with RPMI medium supplemented with 10% FBS, 1% Penicillin-Streptomycin and 1%L-Glutamine.
Fixation
Paraformaldehyde
Other product details
Incubation time
30 minute(s) · Temperature: 23°C · Diluent: Cell Staining Buffer (MaxPar)
Dilution
1/100
Secondary antibody
Secondary antibody
None used
Additional data
Additional Notes
The antibody was conjugated to 158 Gadolinium (Fluidigm) using the standard protocol for antibody conjugation from Fluidigm (file:///Users/odaheleneckfagerholt/Downloads/PRD002+Lanthanide+Labelling+of+Antibodies+V8%20(1).pdf)
HL60 cells and NB4 cells were fixed using 4%PFA, permeabilized using 90% methanol, and frozen at -80C until staining. Cells were thawed on ice. The cells were separately stained with Mdm2 antibody diluted to 1:100 using the protocol provided by Fluidigm (file:///Users/odaheleneckfagerholt/Downloads/PRD016+Phospho+Staining+Protocol+V3%20(3).pdf - starting at step 12). Samples were run on a Helios mass cytometer.
Referring to proteinatlas.org regarding expression of Mdm2 in HL60 and NB4 cell lines.
http://www.proteinatlas.org/ENSG00000135679-MDM2/cell
Abcam response
We advise to include an isotype control to validate the positive signal in NB4 and HL60 cells.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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提交于 May 02 2017