Flow Cytometry abreview for Anti-BrdU antibody [BU1/75 (ICR1)]

Excellent
Abreviews
Application
Flow Cytometry
Sample
Human Cell (Hela)
Specification
Hela
Preparation
Cell harvesting/tissue preparation method: trypsin/EDTA
Sample buffer: 0.5% Tween 20 (v/v) plus 1% BSA in PBSA
Fixation
70% Ethanol
Permeabilization
No
Gating Strategy
Based on forward and side scatter, cells were gated into the region used for analysis. This was done by applying a large circle to accommodate the normal cells in an untreated control.

Other product details

Concentration
0.1 µg/cells
Incubation time
30 minute(s) · Temperature: 25°C · Diluent: 0.5% Tween 20 (v/v) plus 1% BSA in PBSA

Secondary antibody

Name
Non-Abcam antibody was used: goat anti-rat AF 488 (H+L)
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Dilution
1/500

Additional data

PubMed ID
http://www.ncbi.nlm.nih.gov/pubmed/21896658: View
Additional Notes
Cells were incubated with 10 µM BrdU for 30 min prior to being harvested with 1 x trypsin-EDTA, washed twice in PBS containing 1%BSA, and fixed in 70% ethanol (added drop-wise) for at least 30 minutes on ice.
Once fixed, pellets were acid denatured with HCl/Triton X100 for 30 min at room temperature and then neutralised with sodium tetraborate.
Pelleted cells were re-suspended in Tween/BSA/PBS to which primary antibody (Abcam cat#6326) was then added (0.1µg) and incubated for 30 min at room temperature. Secondary anti-Rat-AF488 was used at 1:500 and incubated for 30 at room temperature in the dark.
Cells were pelleted once more and resuspended in PBS containing 5 µg/mL propidium iodide.
Analysis on a Cyan flow cytometer

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Submitted Apr 16 2012

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