Application
Flow Cytometry
Sample
Rat Cell (Brain)
Permeabilization
Yes - 0.25% Triton X-100
Gating Strategy
a single cells population
Specification
Brain
Fixation
Paraformaldehyde
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: overnight
Dilution
1/100
Secondary antibody
Dilution
1/200
Name
Non-Abcam antibody was used: Goat anti-Rabbit IgG (H+L) Cross-Adsorbed
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor 488
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor 488
Additional data
Additional Notes
The samples were incubated with secondary antibodies for 2 hours at room temperature, in the dark with rocking.
IgG ab37416 was used (green on the figure).
A single-cell suspension was prepared by combined a mechanic and enzymatic process.
The tissue was placed to enzymatic digestion solution such as Liberase TM Research Grade (Roche) at the concentration of 1.25 mg/ml for 1.5 hour at 37 ¯C in the thermomixer at 500 g. The digestion process was stopped by PBS+5% BSA. The samples were sieved through 100 ´m cell strainer to remove undigested cell clumps and separate single cells. The various cells from brain tissue were resuspended in PBS+5% FBS, carefully layered on 7.5 ml of Ficoll-Paque PLUS and centrifuge for 30 min at 400 g, room temperature. The interphase containing viable cells were transferred into a clean tube. In order to remove Ficoll-Paque PLUS the samples were washed by PBS+5% FBS and PBS.
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.
MS. Aneta Radziwon-Balicka
Verified customer
投稿 Sep 13 2017