TITLE: Measurement of pancreatic islet beta-cell proliferation by flow cytometry
AUTHORS: Julien  Ghislain, Vincent  Poitout

[DESCRIPTION]
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]
SECTION: Islet dispersion and fixation
1. Dispersing islets into single cells
Wash approximately 100-200 islets two times with ice cold PBS containing 2 millimolar (mM) EDTA (pH 8) in a 1.5 ml microcentrifuge tube using a microcentrifuge at 339 x g, 3 min, 4 °C. 
Keeping the tubes on ice add 200 µL-300 µL 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 10 min.
Return the tubes to ice and add an equal volume of islet media (eg.10% FBS in RPMI 1640), mix and centrifuge at 339 x g, 4 min, 4 °C.
SECTION: Islet dispersion and fixation
2. Live/Dead staining and fixation
Discard the supernatant and wash the cells once with PBS and suspend in 1 mL of PBS onon ice.
Add 2 µL (for human islets) or 1 µL (for rat and mouse islets) of the reconstituted Aqua LIVE/DEAD fluorescent reactive dye, mix and incubate on ice for 30 min (20 min for rat or mouse islets), protected from light.
Centrifuge at 339 x g, 4 min, 4 °C and wash once with 1 % (v/v) BSA in PBS on ice .
Suspend the cell pellet and add 100 µL of Click-iT™ plus EdU kit fixative (Component D), mix and incubate for 15 min at 37 Room temperature protected from light. 
Add 1 mL of 1 % (v/v) BSA in PBS on ice , centrifuge as above and wash with 1 mL 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 on ice.)
SECTION: Staining and flow cytometry
3. Click-iT™ plus EdU reaction
Centrifuge as above and suspend the cell pellet in 50 µLClick-iT™ plus EdU kit perm/wash reagent and mix. Incubate for 10 min 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 37 Room temperature and use within 15 min.

    2 rxn                                   5 rxn
875 µL                2.188 mL                   PBS
20 µL                    50 µL                         Component F
5 µL                      12.5 µL                     488-dye azide 
100 µL                 250 µL                       1X Buffer additive 

Add 450 µL of Click-iT™ plus EdU kit reaction cocktail to each tube, mix and incubate for 30 min at 37 Room temperature, protected from light.
Centrifuge and wash the cells once with 1 mL of Click-iT™ plus EdU kit perm/wash reagent.
SECTION: Staining and flow cytometry
4. Antibody staining
Centrifuge and remove the supernatant. Dislodge the cell pellet and resuspend in 50 µL of Click-iT™ plus EdU kit perm/wash reagent containing 2 µL of Alexa Fluor® 647 Mouse Anti-Insulin and incubate for 20 min at 37 Room temperature in the dark.
Wash twice with 1 mL Click-iT™ plus EdU kit perm/wash reagent and suspend in 200 µL of  1 % (v/v) BSA in PBS.
SECTION: Staining and flow cytometry
5. 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.