May 02, 2022

Public workspaceMeasurement of pancreatic islet beta-cell proliferation by flow cytometry

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldz8d9v5b/v1
Measurement of pancreatic islet beta-cell proliferation by flow cytometry
  • Julien Ghislain1,
  • Vincent Poitout2
  • 1Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada.;
  • 2Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada.
Julien Ghislain
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldz8d9v5b/v1
Protocol Citation: Julien Ghislain, Vincent Poitout 2022. Measurement of pancreatic islet beta-cell proliferation by flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldz8d9v5b/v1
Manuscript citation:
Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans. Maachi H, Ghislain J, Tremblay C, Poitout V. Sci Rep. 2021 May 28;11(1):11283. doi: 10.1038/s41598-021-90643-3. PMID: 34050242.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 23, 2022
Last Modified: May 02, 2022
Protocol Integer ID: 59811
Keywords: flow cytometry, pancreatic islet, beta-cell, proliferation, measurement of pancreatic islet beta, pancreatic islet beta, other islet cells type, cell proliferation by flow cytometry, flow cytometry assay kit, pancreatic beta, suitable for islet, islet, corresponding total insulin, insulin over the corresponding total insulin, cell proliferation, insulin, proliferation marker, proliferating beta, appropriate antibody, antibody, apoptosi
Funders Acknowledgements:
CIHR
Grant ID: 0406014428
NIH
Grant ID: 5R01DK058096-15
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.
Image Attribution
Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans. Maachi H, Ghislain J, Tremblay C, Poitout V. Sci Rep. 2021 May 28;11(1):11283. doi: 10.1038/s41598-021-90643-3. PMID: 34050242.
Materials
ReagentPhosphate Buffered Saline 10x (solution)Bio Basic Inc.Catalog #PD8117
ReagentEDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
ReagentStemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
ReagentRPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
ReagentFBSInvitrogen - Thermo Fisher
ReagentLIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitationThermo FisherCatalog #L34965
ReagentClick-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay KitThermo FisherCatalog #C10632
ReagentBSA fraction VGibco - Thermo Fisher ScientificCatalog #15260037
ReagentAlexa Fluor® 647 Mouse Anti-InsulinBecton Dickinson (BD)Catalog #565689

Troubleshooting
Islet dispersion and fixation
3m
Dispersing islets into single cells
Wash approximately 100-200 islets two times with ice cold PBS containing Concentration2 millimolar (mM) EDTA (Ph8 ) in a 1.5 ml microcentrifuge tube using a microcentrifuge at Centrifigation339 x g, 4°C, 00:03:00 .
Keeping the tubes on ice add Amount200 µL -Amount300 µL of ice cold Accutase solution.
Transfer the tubes to a water bath at Temperature37 °C and gently mix the islet suspension using a 1 ml pipette tip during a maximum of Duration00:10:00 .
Return the tubes to ice and add an equal volume of islet media (eg.10% FBS in RPMI 1640), mix and centrifuge at Centrifigation339 x g, 4°C, 00:04:00 .
17m
Live/Dead staining and fixation
Discard the supernatant and wash the cells once with PBS and suspend in Amount1 mL of PBS onTemperatureOn ice .
Add Amount2 µL (for human islets) or Amount1 µL (for rat and mouse islets) of the reconstituted Aqua LIVE/DEAD fluorescent reactive dye, mix and incubate TemperatureOn ice for Duration00:30:00 (Duration00:20:00 for rat or mouse islets), protected from light.
Centrifuge at Centrifigation339 x g, 4°C, 00:04:00 and wash once with Concentration1 % (v/v) BSA in PBS TemperatureOn ice .
Suspend the cell pellet and add Amount100 µL of Click-iT™ plus EdU kit fixative (Component D), mix and incubate for Duration00:15:00 at TemperatureRoom temperature protected from light.
Add Amount1 mL of Concentration1 % (v/v) BSA in PBS TemperatureOn ice , centrifuge as above and wash with Amount1 mL Click-iT™ plus EdU kit perm/wash reagent. (To store for up to one week at Temperature4 °C , perform instead 2 washes with Concentration1 % (v/v) BSA in PBS TemperatureOn ice .)
1h 9m
Staining and flow cytometry
55m
Click-iT™ plus EdU reaction
Centrifuge as above and suspend the cell pellet in Amount50 µL Click-iT™ plus EdU kit perm/wash reagent and mix. Incubate for Duration00:10:00 Temperature37 °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 TemperatureRoom temperature and use within Duration00:15:00 .

2 rxn 5 rxn
Amount875 µL Amount2.188 mL PBS
Amount20 µL Amount50 µL Component F
Amount5 µL Amount12.5 µL 488-dye azide
Amount100 µL Amount250 µL 1X Buffer additive

Add Amount450 µL of Click-iT™ plus EdU kit reaction cocktail to each tube, mix and incubate for Duration00:30:00 at TemperatureRoom temperature , protected from light.
Centrifuge and wash the cells once with Amount1 mL of Click-iT™ plus EdU kit perm/wash reagent.
55m
Antibody staining
Centrifuge and remove the supernatant. Dislodge the cell pellet and resuspend in Amount50 µL of Click-iT™ plus EdU kit perm/wash reagent containing Amount2 µL of Alexa Fluor® 647 Mouse Anti-Insulin and incubate for Duration00:20:00 at TemperatureRoom temperature in the dark.
Wash twice with Amount1 mL Click-iT™ plus EdU kit perm/wash reagent and suspend in Amount200 µL of Concentration1 % (v/v) BSA in PBS.
20m
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.