May 02, 2022
Measurement of pancreatic islet beta-cell proliferation by flow cytometry
- 1Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada.;
- 2Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada.
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
Funders Acknowledgement:
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
Phosphate Buffered Saline 10x (solution)Bio Basic Inc.Catalog #PD8117
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
RPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
FBSInvitrogen - Thermo Fisher
LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitationThermo FisherCatalog #L34965
Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay KitThermo FisherCatalog #C10632
BSA fraction VGibco - Thermo FischerCatalog #15260037
Alexa Fluor® 647 Mouse Anti-InsulinBD BiosciencesCatalog #565689
Islet dispersion and fixation
Islet dispersion and fixation
3m
3m
Dispersing islets into single cells
Wash approximately 100-200 islets two times with ice cold PBS containing 2 millimolar (mM) EDTA (8 ) in a 1.5 ml microcentrifuge tube using a microcentrifuge at 339 x g, 4°C, 00:03:00 .
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 00:10:00 .
Return the tubes to ice and add an equal volume of islet media (eg.10% FBS in RPMI 1640), mix and centrifuge at 339 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 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 00:30:00 (00:20:00 for rat or mouse islets), protected from light.
Centrifuge at 339 x g, 4°C, 00:04:00 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 00:15:00 at 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 .)
1h 9m
Staining and flow cytometry
Staining and flow cytometry
55m
55m
Click-iT™ plus EdU reaction
Centrifuge as above and suspend the cell pellet in 50 µL Click-iT™ plus EdU kit perm/wash reagent and mix. Incubate for 00:10:00 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 Room temperature and use within 00:15:00 .
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 00:30:00 at Room temperature , protected from light.
Centrifuge and wash the cells once with 1 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 50 µL of Click-iT™ plus EdU kit perm/wash reagent containing 2 µL of Alexa Fluor® 647 Mouse Anti-Insulin and incubate for 00:20:00 at 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.
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.