Protocol for high-throughput viability screening and gene expression of human EndoC-βH5 β cells and pancreatic islets

Summary

Viability assays allow the assessment of toxicity induced by any treatment of interest. Here, we present a protocol to evaluate cell viability in human EndoC-βH5 β cells and human pancreatic islets using a high-throughput fluorescence viability assay. We describe the steps for cell and islet culture, reagent dilution, viability assay preparation, and performance. The protocol is compatible with RNA isolation and gene expression analysis on the same cells, and we detail procedures for reagent washout and profiling gene expression.

For complete details on the use and execution of this protocol, please refer to Rampazzo Morelli et al.¹

Graphical abstract

Highlights

• •Protocol to measure relative cell viability of EndoC-βH5 β cells and human islets
• •Instructions for culturing EndoC-βH5 cells and human islets for the viability assay
• •Steps for reagent dilution, viability assay setup, and performance
• •Guidance on reagent removal for gene expression analysis from the same cells

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Viability assays allow the assessment of toxicity induced by any treatment of interest. Here, we present a protocol to evaluate cell viability in human EndoC-βH5 β cells and human pancreatic islets using a high-throughput fluorescence viability assay. We describe the steps for cell and islet culture, reagent dilution, viability assay preparation, and performance. The protocol is compatible with RNA isolation and gene expression analysis on the same cells, and we detail procedures for reagent washout and profiling gene expression.

Before you begin

Viability assays are widely used in research to assess how cells respond to specific treatment conditions. Currently, there are several viability assays available that can be classified as dye exclusion assays (e.g. trypan blue), colorimetric assays (e.g. MTT), fluorometric assays (e.g. resazurin-based reagents), luminometric assays (e.g. ATP assay), and flow cytometric assays.² In this protocol, we describe the applicability of PrestoBlue high-sensitivity (HS) viability assay on human beta cells and isolated islets which uses a nontoxic resazurin-based reagent (https://www.thermofisher.com/order/catalog/product/P50200). The principal of the method is based on an irreversible reduction of resazurin to resorufin by live cells. Resorufin is a pink compound which is metabolized by the live cells into the media, leading to a color change of the media that can be detected by fluorescent filters. Viable cells will be constantly reducing resazurin to resorufin, which leads to increased fluorescence.²

Human donor islets are an important and costly resource to the islet biology and diabetes research community. In vitro human beta cell models such as EndoC-βH5 also provide a valuable tool to study human beta cell physiology and function as a complementary system to donor islets. Experiment workflows using these resources greatly benefit from multiplexed assays on the same samples. Islets and EndoC-βH5 cells are non-proliferative and their viability is sensitive to a wide range of insults, necessitating accurate and sensitive viability screening methods. The PrestoBlue HS assay allows for the ability to carry the same islets or cells forward for gene expression studies and therefore provides a powerful alternative to other endpoint viability assays that destroy cells, such as trypan blue staining or the MTT assay.

Institutional permissions

Studies involving de-identified human cadaveric donor pancreatic islets require institutional ethics board review. Studies using de-identified human islets described in this protocol were carried out using research ethics board exemption HS25660 at the University of Manitoba. Human islets for research in our protocol were provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope, NIH Grant No. U24DK098085.

Culture of EndoC-βH5 β cells or human pancreatic islets

Timing: Variable

• 1.
  Seed EndoC-βH5 cells or human islets accordingly.

  • a.
    Before thawing EndoC-βH5 cells, TPP plates must be coated with βCOAT gel matrix.

    • i.
      Keep the plate in an incubator at 37°C and 5% CO₂ for at least 1 h.
    • ii.
      Thaw and seed EndoC-βH5 cells following the instructions described in the user guide, including recommendations on number of cells seeded per well and density (e.g. 3.75 x 10⁵ cells/mL/well in a 12-well plate).
    • iii.
      After incubation, remove the media and replace with fresh ULTIβ1 media. Cells must be cultured in an incubator at 37°C and 5% CO₂ for 48 h before carrying out the experiments.

      CRITICAL: While seeding, make sure EndoC-βH5 cells are uniformly distributed in the wells to avoid cells overlapping, consequently, not attaching properly.

      Note: All the steps and details on EndoC-βH5 cells culturing are available in the user guide provided by Human Cell Design. See the link for the user guide on key resources table below.

  • b.
    Upon receipt, the tube containing human islets can be centrifuged to collect islets (150 x g for 2 min at 20°C–25°C).

    • i.
      Remove the supernatant carefully with a serological and/or micropipettor. To avoid losing islets during this process, the use of a vacuum system or pouring out the supernatant is not recommended.
    • ii.
      Add 15 mL human islet media into the tube, followed by transferring all the content into a petri dish.
    • iii.
      Islets must rest for 18–24 h in an incubator at 37°C and 5% CO₂ before being hand-picked into a culture plate for the experiments.

      CRITICAL: After processing the islets upon receipt, it is important to check them under an optical microscope to make sure islets are in good condition to carry out the experiments. Additionally, for human islet preparations containing over 5,000 islet equivalents (IEQ), it is recommended to split all the contents into two or more petri dishes with 15 mL of media each to avoid over-crowding.

      Note: It is recommended to use low-retention micropipette tips to pick up the islets as well as to use non-treated petri dishes and culture plates.

      Note: For EndoC-βH5 cells, viability assay is optimized using 1 x 10⁵ cells per well in a 96-well plate or 1.85 x 10⁵ per well in a 24-well plate. For human islets, viability assay is optimized using 10-15 islets per well in a non-treated 24-well tissue culture plate.

• 2.Perform the treatment of interest on cells or islets.

Note: Treatment time point (e.g. 24 h) is determined according to experimental design planned.

Note: It is recommended to include a positive control treatment, such as ≥0.5 μM staurosporine (pan-kinase inhibitor) that induces apoptosis.

PrestoBlue HS cell viability reagent preparation

Timing: <30 min

• 3.
  Warm up the viability reagent at 20°C–25°C before preparing the working solution.

  • a.
    Prepare working space and materials accordingly in the meantime.
• 4.
  Prepare a working solution for the viability reagent.

  • a.
    Calculate total volume needed based on volume added per well.

    • i.
      0.2 mL/well for 96-well plates.
    • ii.
      0.5 mL/well for 24-well plates.
    • iii.
      1 mL/well for 12-well plates.
  • b.
    Dilute viability reagent from the stock at 10x using cell or islet media to prepare a working solution at 1x.
  • c.
    Protect the working solution from light.

Note: Viability reagent (stock) is stable for six months upon receipt when stored at 2°C–8°C and protected from light. The working solution must be prepared fresh to use.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Human donor islets	IIDP	RRID:SAMN37735327 RRID:SAMN40709610
EndoC-βH5 human β cells	Human Cell Design	https://www.humancelldesign.com/download-user-guide-endoc-bh5
Chemicals, peptides, and recombinant proteins
RPMI 1640 medium, no glucose (1x)	Gibco	Cat# 11879020
Fetal bovine serum (FBS)	Gibco	Cat# A5256701
Antibiotic-antimycotic (100x)	Gibco	Cat# 15240096
D-(+)-glucose, 1 M aq. soln., sterile-filtered	Thermo Scientific	Cat# J60067.AK
DPBS, no calcium, no magnesium (1x)	Gibco	Cat# 14190144
Dimethyl sulfoxide, Bioreagent (DMSO)	Thermo Scientific	Cat# J66650.AD
Bleomycin sulfate	MedChemExpress	Cat# HY-17565
Staurosporine	Cayman Chemical	Cat# 81590
Trypsin-EDTA (0.05%), phenol red	Gibco	Cat# 25300054
TRI Reagent	Zymo Research	Cat# R2050-1-200
Critical commercial assays
PrestoBlue HS cell viability reagent (10x)	Invitrogen	Cat# P50200
Direct-zol RNA MicroPrep	Zymo Research	Cat# R2062
RNeasy Plus micro kit	QIAGEN	Cat# 74034
LunaScript RT SuperMix kit	New England Biolabs	Cat# E3010
Luna universal qPCR master mix	New England Biolabs	Cat# M3003
Experimental models: Cell lines
EndoC-βH5 human beta cells	Human Cell Design	Cat# BH5-WT
ULTIβ1 serum free medium for EndoC cells	Human Cell Design	Cat# UB1-100-BSA
βCoat coating matrix for EndoC cells	Human Cell Design	Ca # BC-120
TPP plates (24-well, flat bottom) for culturing EndoC cells	MIDSCI	Cat# TP92024
Oligonucleotides
Primer: human ACTB (β-actin) FWD: 5′-AGC TAC GAG CTG CCT GAC-3′ REV: 5′ AAG GTA GTT TCG TGG ATG C-3′	Integrated DNA Technologies	Brawerman et al.3
Primer: human GAPDH FWD: 5′- TTG AGG TCA ATG AAG GGG TC-3′ REV: 5′- GAA GGT GAA GGT CGG AGT CA3′	Integrated DNA Technologies	Brawerman et al.3
Software and algorithms
BioTek Gen5	Agilent	https://www.agilent.com/en/support/biotek-software-releases
GraphPad Prism	Dotmatics	https://www.graphpad.com
Other
BioTek Cytation 5	Agilent	N/A

DPBS, Dulbecco’s Phosphate Buffered Saline; EDTA, Ethylenediaminetetraacetic acid; TPP, Techno Plastic Products; FWD, Forward; REV, Reverse; GAPDH, Glyceraldeyde-3-phosphate dehydrogenase.

Materials and equipment

Human islet media

Reagent	Final concentration	Amount
RPMI 1640 (1x)	90%	450 mL
FBS	10%	50 mL
Antibiotic-Antimycotic (100x)	1x	5 mL
D-glucose (1 M)	5.5 mM	2.75 mL
Total	N/A	507.75 mL

Store at 4°C for up to 4 weeks.

Step-by-step method details

Assay preparation

Timing: <1 h

After diluting the working solution for the viability reagent, the plate containing cells or islets previously treated needs to be prepared to perform the assay.

• 1.Remove media from cells or islets.

Note: Islets generally do not attach to the plate, for this reason, the media needs to be aspirated carefully. It is recommended to use a micropipettor to perform this step and not disturb the islets.

• 2.
  Add the working solution to the wells.

  • a.
    After adding the viability reagent, protect the plate from light while waiting to start the assay.

Note: If work involves many samples, to prevent cells or islets drying out while performing these steps, it is recommended to remove the media and add the reagent into a smaller group of wells per time.

Viability assay

Timing: <3 h

After preparing the plate, the viability assay is ready to be performed. The equipment used for this assay is designed to provide a favorable environment, including temperature at 37°C, 5% CO₂, and humidity to keep cells or islets incubating to carry out the readings (Figure 1A).

• 3.
  Set up the protocol to perform the viability assay.

  • a.
    Select a respective layout for the culture plate used.
  • b.
    Select the fluorescence wavelengths.

    • i.
      Excitation of 560 nm.
    • ii.
      Emission of 590 nm.
  • c.
    Set up readings at the following incubation time points.

    • i.
      Baseline (Reading 1), 10 min incubation (Reading 2), 30 min incubation (Reading 3), 60 min incubation (Reading 4), 60 min incubation (Reading 5) (Figure 1A).
• 4.Start the viability assay.

CRITICAL: Fluorescence signals should increase at every reading after the incubation time points.

Note: Optimized timing to carry out all the readings is 2 h 40 min.

Figure 1.

Flowcharts

(A) Optimized incubation time points to perform the viability assay.

(B) Steps carried out in EndoC-βH5 human beta cells and pancreatic human islets from two independent donors. Donor 1 islets underwent the viability assay prior to RNA isolation whereas Donor 2 islets did not.

Reagent removal and sample harvesting

Timing: Variable

The viability reagent used on this assay does not require cell lysis, allowing cells or islets to be harvested and used for RNA extraction and RT-qPCR after removing the viability reagent. We show in this protocol that the quality of RNA extracted from human islets after performing the viability assay is comparable with RNA extracted from islets without performing the viability assay (Figure 1B). See more details on the expected outcomes section below.

• 5.
  Remove the viability reagent from cells or islets.

  • a.
    Cells: Carefully aspirate viability reagent from the wells.

    • i.
      Wash cells twice with 1x DPBS.
  • b.
    Islets: collect all the contents from the wells and transfer into a microtube.

    • i.
      Centrifuge microtube at 150 x g for 2 min at 20°C–25°C.
    • ii.
      Carefully aspirate supernatant containing viability reagent.
    • iii.
      Wash islets twice with 0.2–0.5 mL of 1x DPBS, repeating the centrifuge step and aspirating the supernatant after each wash.
• 6.
  Prepare cells or islets for RNA extraction.

  • a.
    Cells: After washing, add enough 0.05% Trypsin-EDTA to cover the cells (e.g. 0.1 mL for 96-well plates, 0.25 mL for 24-well plates) and keep the plate in an incubator at 37°C and 5% CO₂ for 5 min.

    • i.
      Quench Trypsin-EDTA by adding equal volume (e.g. 0.25 mL for 24-well plates) of a solution containing 90% DPBS and 10% FBS.
    • ii.
      Collect all the content from the wells transferring into a microtube and centrifuge at 500 x g for 5 min at 20°C–25°C.
    • iii.
      Aspirate supernatant and add lysis buffer for RNA extraction from RNeasy Plus Micro Kit.
  • b.
    Islets: After washing, add TRI Reagent (Trizol) for RNA extraction.

Note: After adding respective lysis reagents to cells or islets, samples can be frozen at −80°C or processed immediately if desired.

• 7.
  Perform the RNA extraction according to the appropriate protocol.

  • a.
    RNeasy Plus Micro Kit is recommended for EndoC-βH5 cells.
  • b.
    Direct-Zol RNA MicroPrep is recommended for islets.
  • c.
    Quantify isolated total RNA using Nanodrop 2000 spectrophotometer or similar device.

    • i.
      Use equal amounts of extracted total RNA for cDNA synthesis using LunaScript RT SuperMix Kit or similar.
    • ii.
      Carry out quantitative PCR (qPCR) using Luna Universal qPCR Master Mix for gene expression.

Expected outcomes

After the baseline reading, it is expected that a continuous increase in fluorescence will occur while the samples incubate. Usually, the fluorescence intensity does not change after 10 minutes of incubation, but within 30 minutes it is possible to start seeing an increase in the signals. Increasing fluorescence intensity is observed with longer periods of incubation, as demonstrated in the last two readings followed by 60-minute of incubation each (Figures 2A and 2B). Small molecule agents that can trigger cell death, such as staurosporine and bleomycin can be used as positive controls in the assay compared with vehicle only negative controls. At the end, samples with fewer viable cells will have lower fluorescence intensity compared to a negative vehicle only or untreated control. This is observed in EndoC- βH5 cells treated with staurosporine (0.5 μM) as a positive control for cell death in comparison to DMSO vehicle treatment as a negative control for 72 h (Figure 2A), as well as in human islets from donor 1 treated with bleomycin (50 μM) to induce DNA damage for 48 h and kept in culture for 5 more days after removing the drug, compared to DMSO vehicle negative control (Figure 2B).

Figure 2.

High-throughput fluorescence viability assay and RT-qPCR analysis in EndoC-βH5 human beta cells and pancreatic human islets

Plotting of relative background normalized fluorescence units (RFU) from (A) EndoC-βH5 cells after treatment with 0.5 μM staurosporine as a positive control for cell death or 0.05% DMSO as a negative control, for 72 h and (B) human islets from donor 1 (54 years, female, BMI 23.3) after treatment with 50 μM bleomycin for 48 h, drug washout and culture in islet media for 5 days. Data are mean ± SD from n = 3 biological replicates. ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-way ANOVAs or two-tailed T-tests.

(C) Table with Nanodrop 2000 spectrophotometer concentrations (ng/μL) and 260/280 ratio for extracted RNA from EndoC-βH5 cells and human islets from donor 1 after performing the viability assay, as well as for human islets from donor 2 (55 years, female, BMI 28.6) without performing the viability assay. Sufficient total RNA was recovered from each sample to generate 100 or 200 ng of cDNA.

(D) Cycle threshold (C_t) values for GAPDH housekeeping gene in EndoC-βH5 cells after performing the viability assay on cells treated with 50 μM bleomycin for 48 h and drug washout and culture for 5 days. Note RNA recovery from staurosporine-treated EndoCβH5 cells (A) was insufficient for qPCR. Data are mean ± SD from n = 4 biological replicates. ns = not significant, two-tailed T-test.

(E) Comparison between C_t values for GAPDH and ACTB (Beta-actin) housekeeping genes in human islets from donor 1 and donor 2. Data are represented as mean ± SD. Data are mean ± SD from n = 4 biological replicates. ns = not significant, two-way ANOVAs.

In order to recover sufficient total RNA after the viability assay for RT-qPCR, it is also important to consider the number of cells or islets seeded for the experiment. EndoC- βH5 cells showed good results using a minimum of 1.85 x 10⁵ cells. Using around 10-15 medium-large-sized islets (on average ∼1,000 cells per islet) it is possible to recover a sufficient amount and quality of RNA for cDNA (100 ng) synthesis after the viability assay (human islets from donor 1). Importantly, the quality of this RNA is comparable to RNA extracted from 30 human islets of similar size that did not undergo the viability assay (human islets from donor 2) and generated 200 ng of cDNA (Figure 2C). Thus, the amount and quality of RNA isolated will depend upon how many viable cells remain in the cultures after treatment, regardless whether the viability assay is performed or not. Notably, the viability assay does not interfere with qPCR results, as demonstrated by the cycle threshold (C_t) from housekeeping genes (GAPDH and ACTB) in EndoC-βH5 cells (Figure 2D) and in human islets from donor 1 (that underwent the viability assay), as compared with human islets from donor 2 (Figure 2E) that contained more islets and did not undergo the viability assay (Figure 1B). The protocol for bleomycin treatment of human islets to induce DNA damage was the same for both donors.¹

Quantification and statistical analysis

Quantification will consist of relative fluorescence units (RFU) normalized by subtracting fluorescence values obtained in the baseline reading (Reading 1) from fluorescence values obtained in the final reading (Reading 5). For final plotting, an appropriate statistical analysis must be selected according to experimental design. Two-way ANOVA with Tukey’s post hoc was used to analyze the viability between treatment conditions in EndoC-βH5 cells and human islets from donor 1 at each reading. After normalizing the data (background subtraction), a two-tailed t-test was applied. C_t values from housekeeping genes in the treatment conditions were plotted using two-tailed t-test for EndoC-βH5 cells and two-way ANOVA with Tukey’s post hoc for human islets to compare values between the donors. Data are represented as mean ± SD of n = 3 or 4 biological replicates. Statistical analyses were performed on GraphPad Prism and results were considered significant at p < 0.05.

Limitations

The main benefits of this assay are its ease of use and the compatibility with downstream RNA isolation in order to maximize the experiments performed on the same samples. The assay can rapidly screen relative differences in human islet and EndoC-βH5 cell viability across a large number of samples and hence is faster to perform than lower-throughput approaches. However, its main limitation is that it cannot determine absolute viable cell numbers (e.g. the percent of live cells in the well). Subtle changes in viable cell number are more difficult to detect with this assay due to the fact that the assay measures the fluorescence readout of each whole well of cells or islets, not viability on a single cell level. Another limitation is inherent in the fact that it uses a reducing reaction as the readout. Chemical treatments or agents that interfere with the redox balance of the cells or islets may interfere with the accuracy of the result. Due to these considerations, it is highly recommended to independently corroborate or verify changes in cell or islet viability found during initial screening with this assay using an orthogonal approach, such as a dye-exclusion-based assay that can determine viable cell percentage in the culture. In addition, a methodological limitation is the use of a specific plate reader (BioTek Cytation 5 or higher, Agilent) to perform the assay. This is recommended because this equipment has the advantage of simultaneously incubating the samples and carrying out the readings. The equipment is designed to maintain temperature at 37°C and at 5% CO₂, which requires connection to CO₂ cylinder, and humidity is controlled with a small water container attached to the plate tray. However, if this equipment or similar is not available, it is possible to keep the samples in a standard CO₂-controlled tissue culture incubator and perform the readings at each interval in a regular fluorescence plate reader.

Troubleshooting

Problem 1

Lack of increase in fluorescence signals after the final time point (related to step 4).

Potential solution

• •Insufficient incubation time. Cells or islets may need longer time of incubation. Ensure that the signal increases in the negative control healthy cells or islets at the reading 5 time point as compared with the baseline.
• •Insufficient live islets or cells in the wells. If the treatment(s) tested killed the vast majority of cells (>90%) then no changes in fluorescence will occur.
• •Use a greater number of cells or islets per well. Typically, at least 10-15 medium-large sized human islets (∼1,000 cells/islet) are sufficient to generate meaningful fluorescence data. A minimum of 1.85 x 10⁵ cells per well of a 24-well plate (or scaled appropriately for larger or smaller plates) should be used for viability and RNA isolation with EndoC-βH5.
• •Use a positive control cell line. The assay can be easily tested using any number of transformed human cell lines as a control (E.g. 293T, HeLa, etc.), which should show the expected increase.

Problem 2

Insufficient or no RNA recovered after performing the viability assay (related to step 7).

Potential solution

• •Insufficient washing with DPBS prior to RNA isolation. Ensure cells or islets were washed properly with DPBS to completely remove the viability reagent. Insufficient washing of cells or islets reduces yields of purified total RNA.
• •Insufficient cells or islets used (see problem #1 above). Increase number of islets or cells plated per well prior to assay.
• •Viability of cells was too low. If there is no increase in fluorescence signal during the assay, it is possible that there are not enough live cells in the well for RNA isolation.
• •Incorrect RNA isolation kit used. Use optimal kit for sample type. Trizol-based kits work best on islets, while the RNeasy plus kit works best for EndoC-βH5.

Problem 3

High variability in fluorescence data between similarly treated wells in viability assay (related to step 4).

Potential solution

• •Differences between wells in EndoC-βH5 seeding density, or number of human islets per well. Ensure cells were counted and seeded at the appropriate density. Ensure each well contains the same number of similarly sized islets.
• •Insufficient number of biological replicates per treatment condition. Depending on the treatment, the viability data may be quite variable. While n=4 biological replicates are often suitable, this may need to be increased to n=5 or 6 replicates to determine statistically significant differences between treatments.
• •Incorrect plate type for fluorescence readings. Ensure the correct plate type appropriate for your fluorescence detection system is used. Clear tissue culture plates are recommended, while opaque plates should be avoided.

Problem 4

High variability in RNA recovery or purity between similarly treated wells after viability assay (related to step 7).

Potential solution

• •Differences between wells in EndoC-βH5 seeding density, or number of human islets per well. Ensure cells were counted and seeded at the appropriate density. Ensure each well contains the same number of similarly sized islets.
• •Insufficient washing prior to RNA isolation. Ensure the cells and islets are washed thoroughly with DPBS before recovery for RNA isolation. The washing step is critical for removal of excess dye prior to Trizol-mediated RNA extraction.
• •Incorrect RNA isolation procedure or kit used. For best results following viability assay, it is recommended to use a Trizol-based extraction kit for human islets and the RNeasy plus kit for EndoC-βH5 cells.

Problem 5

Treatment has no effect on viability as measured by assay (related to step 4).

Potential solution

• •Lack of positive control to verify loss of viability. It is recommended to include a positive control treatment that is known to lead to loss of cell or islet viability. We suggest the use of staurosporine or bleomycin, which each lead to reductions in viability as a positive control (Figures 2A and 2B).
• •No alternative viability assay used to compare. Based on the limitations of this viability assay, there could be changes in cell viability which are not reflected at the level of the reducing reaction using this reagent. A second complementary assay is generally recommended to independently confirm the result.
• •PrestoBlue HS reagent is expired or no longer functional. Confirm that the product is within its shelf-life and was stored correctly at 4°C. Do not use expired reagent for viability assays.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Peter J. Thompson (peter.thompson@umanitoba.ca).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Nayara Rampazzo Morelli (nayara.rampazzomorelli@umanitoba.ca).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets or code.

Acknowledgments

N.R.M. was supported by a Canadian Islet Research and Training Network-Breakthrough T1D postdoctoral training award. The laboratory of P.J.T. was supported by an End Diabetes 2022 award from Diabetes Canada (OG-3-22-5694-PT), the 2023 Manitoba Medical Service Foundation Allen Rouse Basic Science Career Development Award, a Canadian Institutes of Health Research (CIHR) project grant (PJT-479641), team grants from CIHR (485691 and 485915), Breakthrough T1D (4-SRA-2023-1182-S-N and 4-SRA-2023-1184-S-N), the Canada Foundation for Innovation John R. Evans Leaders Fund (43168), and an NSERC Discovery grant (RGPIN-2024-05471). Figures were prepared using BioRender.

Author contributions

N.R.M. and P.J.T. conceived the study and designed the protocol. N.R.M. carried out the study and tested the protocol. N.R.M. and P.J.T. wrote and edited the manuscript together.

Declaration of interests

The authors declare no competing interests.