Measurement of pancreatic islet beta-cell proliferation by flow cytometry
Julien Ghislain, Vincent Poitout
Abstract
This protocol describes the steps to assess pancreatic beta-cell proliferation by flow cytometry. It is suitable for islets isolated from both rodents and humans. We routinely apply this protocol to quantify total and proliferating beta-cells but, with the appropriate antibody-fluorophore conjugates, can be easily adapted to assess other islet cells types (eg. alpha and delta) and markers (eg. apoptosis). Briefly, islets labelled with the proliferation marker EdU are dispersed, dead cells labeled and fixed. EdU is detected using the Click-iT Plus EdU Flow Cytometry Assay Kit and beta-cells immunostained with fluorophore-coupled anti-insulin antibodies. Beta-cell proliferation is then determined by flow cytometry by calculating the percentage of double-positive cells for EdU and insulin over the corresponding total insulin-positive cell population.
Steps
Islet dispersion and fixation
Dispersing islets into single cells
Wash approximately 100-200 islets two times with ice cold PBS containing 2millimolar (mM) EDTA (8) in a 1.5 ml microcentrifuge tube using a microcentrifuge at 339x g,4°C.
Keeping the tubes on ice add 200µL-300µL of ice cold Accutase solution.
Transfer the tubes to a water bath at 37°C and gently mix the islet suspension using a 1 ml pipette tip during a maximum of 0h 10m 0s.
Return the tubes to ice and add an equal volume of islet media (eg.10% FBS in RPMI 1640), mix and centrifuge at 339x g,4°C.
Live/Dead staining and fixation
Discard the supernatant and wash the cells once with PBS and suspend in 1mL of PBS on37On ice.
Add 2µL (for human islets) or 1µL (for rat and mouse islets) of the reconstituted Aqua LIVE/DEAD fluorescent reactive dye, mix and incubate 37On ice for 0h 30m 0s (0h 20m 0s for rat or mouse islets), protected from light.
Centrifuge at 339x g,4°C and wash once with 1% (v/v) BSA in PBS 37On ice .
Suspend the cell pellet and add 100µL of Click-iT™ plus EdU kit fixative (Component D), mix and incubate for 0h 15m 0s at 37Room temperature protected from light.
Add 1mL of 1% (v/v) BSA in PBS 37On ice , centrifuge as above and wash with 1mL Click-iT™ plus EdU kit perm/wash reagent. (To store for up to one week at 4°C, perform instead 2 washes with 1% (v/v) BSA in PBS 37On ice.)
Staining and flow cytometry
Click-iT™ plus EdU reaction
Centrifuge as above and suspend the cell pellet in 50µLClick-iT™ plus EdU kit perm/wash reagent and mix. Incubate for 0h 10m 0s 37°C. During the incubation step prepare the Click-iT™ plus EdU kit reaction cocktail.
Start by preparing a 1X Click-iT™ plus EdU kit buffer additive by diluting the 10X stock solution in water.
Then, prepare the Click-iT™ plus EdU kit reaction cocktail at 37Room temperature and use within 0h 15m 0s.
**2 rxn** **5 rxn** _rxn_
875µL 2.188mL PBS
20µL 50µL Component F
5µL 12.5µL 488-dye azide
100µL 250µL 1X Buffer additive
Add 450µL of Click-iT™ plus EdU kit reaction cocktail to each tube, mix and incubate for 0h 30m 0s at 37Room temperature, protected from light.
Centrifuge and wash the cells once with 1mL of Click-iT™ plus EdU kit perm/wash reagent.
Antibody staining
Centrifuge and remove the supernatant. Dislodge the cell pellet and resuspend in 50µL of Click-iT™ plus EdU kit perm/wash reagent containing 2µL of Alexa Fluor® 647 Mouse Anti-Insulin and incubate for 0h 20m 0s at 37Room temperature in the dark.
Wash twice with 1mL Click-iT™ plus EdU kit perm/wash reagent and suspend in 200µL of 1% (v/v) BSA in PBS.
Flow cytometry
Dead-cell stain, EdU and insulin labelled cells are detected using 405-, 488-, 640-nm lasers coupled with 525/50-, 530/30-, 670/14-nm BP filters, respectively, or similar. Proliferation is calculated as the percentage of double-positive EdU+ and Ins+ cells over the total Ins+ cell population.
