Admin: Created page with "==Reagents== Binding Buffer AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488) *Propridium Iodide ==Protocol== ===Binding Buffer=== Prepare binding buffer, for..."

2021-02-01T19:55:24Z

Created page with "==Reagents== *Binding Buffer *AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488) *Propridium Iodide ==Protocol== ===Binding Buffer=== Prepare binding buffer, for..."

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==Reagents==
*Binding Buffer
*AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488)
*Propridium Iodide

==Protocol==
===Binding Buffer===
Prepare binding buffer, for 100 mL:
*238 mg HEPES (10 mM)
*818 mg NaCl (140 mM)
*37 mg CaCl2.2H2O (2.5 mM CaCl)
100 mL ddH2O
pH to 7.4

==Suspension Cells, or for FACS==
#To 100 ul of binding buffer add 1 ug/ml (1 ul of a 100X solution) propridium iodide + 5 ul of AnnexinV conjugate
#Pellet 1x105 cells (suspend by trypsinization or scriping first, if required), wash 1x with binding buffer, then resuspend in the PI/AnnexinV buffer
#Incubate '''in the dark''' at room temperature for 15 min
#Add an additional 400 ul of ice-cold binding buffer, and store samples on-ice for imaging. Image or FACS analyzie as soon as possible.

==For Microscopy==
#Grow cells on coverslips and treat as per your protocol
#Prepare 100 ul of labeling buffer/coverslip: 0.5 ug/ml (0.5 ul of a 100X solution) propridium iodide + 2.5 ul of AnnexinV conjugate
#Using the hanging drop method, place the '''live and unfixed cells''' on a droplet of labeling buffer, at room temperature, for 15 min in the dark.
#*If imaging live, mount coverslip in spaceship and image immeidatly.
#*If fixing/mounting the sample:
##During the incubation, prepare 4% PFA in binding buffer (not in PBS)
##Wash coverslip 1x with binding buffer, then add 4% PFA solution. Incubate 20 min at room temperature.
##Wash coverslip 1x with binding buffer, then mount slide and image as per normal

Apoptosis Detection with AnnexinV and PI - Revision history

Admin: Created page with "==Reagents== *Binding Buffer *AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488) *Propridium Iodide ==Protocol== ===Binding Buffer=== Prepare binding buffer, for..."

Admin: Created page with "==Reagents== Binding Buffer AnnexinV-flourophore conjugate (e.g. ThermoFisher AnnexinV-488) *Propridium Iodide ==Protocol== ===Binding Buffer=== Prepare binding buffer, for..."