TITLE: Human Islet Cell Culture
AUTHORS: Bridget Wagner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Human islet cell culture</div></div>

[STEPS]
?. [Cell-culture formulations]
The HTB-9 bladder carcinoma cell line (ATCC) is cultured in medium containing RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1 x penicillin-streptomycin.
?. [Cell-culture formulations]
Islet culture medium: Islets are cultured in CMRL-1066 supplemented with 10% FBS, 1 x penicillin-streptomycin, and 2mM L-glutamine.
?. [Conditioned medium: Preparation of matrix-coated 384-well plates]
Culture HTB-9 cells in T-175 flasks until 100% confluent, then incubate for an additional 3 days after changing media.
?. [Conditioned medium: Preparation of matrix-coated 384-well plates]
Collect the conditioned HTB-9 supernatant, and filter with 0.22 mm filter and store at -20C until use.
?. [Conditioned medium: Preparation of matrix-coated 384-well plates]
The day before you plan to seed islet cells, coat each well of a 384-well plate (PerkinElmer, CellCarrier-384 Ultra) with 10 mL/well HTB-9 supernatant and incubate overnight at 37C, 5% CO2. If you are short of time, 1-2 h is acceptable.
?. [Conditioned medium: Preparation of matrix-coated 384-well plates]
Wash each well twice with 50 mL/well PBS.
?. [Conditioned medium: Preparation of matrix-coated 384-well plates]
Add 10 mL/well islet culture medium (see above) and return to the incubator in advance of receiving islets.
?. [Conditioned medium: Preparation of matrix-coated 384-well plates]
As an alternative, coated plates filled with 50 mL/well PBS (after the same two washes) can be stored at 4C for up to 1 month.
?. [Islet culture in 384-well plates]
Upon receipt, pellet islets by centrifugation in a 50-mL Falcon tube and wash once with PBS.
?. [Islet culture in 384-well plates]
Dissociate islets in Accutase (5000 IEQ/mL) in a Falcon tube for 20 min at 37C in 5% CO2 incubator, keeping cell clumps down to ~10 cells. After ~10 min, agitate the Falcon tube and return to the incubator.
?. [Islet culture in 384-well plates]
Pipette 5-7 times gently.
?. [Islet culture in 384-well plates]
Add CMRL medium to dilute Accutase (9 mL for every 1 mL Accutase).
?. [Islet culture in 384-well plates]
Resuspend islets in islet culture medium (see above) at a density of 250,000 cells/mL.
?. [Islet culture in 384-well plates]
Dispense 40 mL/well into coated 384-well plates, which will give you 10,000 cells/well. We use the MultiDrop Combi (Thermo Fisher) for semi-automated dispensing.NOTE: this section can be adapted to 96-well format by altering the volumes and cell density. NOTE: this section can be adapted to 96-well format by altering the volumes and cell density.
?. [Cell proliferation]
After incubating for 24hr at 37C, 5% CO2, aspirate medium, leaving 10 mL/well. We use the BioTek ELx-405 automated plate washer for this step. Unless otherwise indicated, we aspirate medium so that 10 mL remains, and then add 40 mL/well to bring the total to 50 mL.
?. [Cell proliferation]
Add 40 mL/well medium containing 12.5 mM EdU (final concentration = 10 mM). Total volume is 50 mL/well.
?. [Cell proliferation]
Treat and incubate according to your experiment. We transfer compounds from compound plate (Abgene 384 PP, V-bottom) to cell plates using the Cybio Well Vario (Analytik Jena). We have found that the final DMSO concentration should remain ≤ 0.3%.
?. [Cell proliferation]
Change the culture medium containing 10 mM EdU (and add compounds as appropriate) every two days.
?. [Cell proliferation]
Aspirate the medium, then add 40 mL/well 4% PFA/PBS (final concentration will be 3.2%) at room temperature for 20 min.
?. [Cell proliferation]
Wash cells twice with PBS.
?. [Cell proliferation]
Permeabilize islet cells with 0.2% TritonX-100/PBS (final concentration) at room temperature for 20min.
?. [Cell proliferation]
Wash twice in PBS.
?. [Cell proliferation]
Stain cells using the Click-iT EdU HCS assays (catalog number C10351, Invitrogen) according to the manufacturer’s protocol.
?. [Cell proliferation]
Block with 2% BSA/PBS at room temperature for 2 hr while gently shaking. No need to wash after this step.
?. [Cell proliferation]
Incubate primary antibody (C-peptide) at 1:100 dilution in 2% BSA/PBS overnight at 4C with gentle shaking.
?. [Cell proliferation]
Wash cells three times with PBS, then once with 2% BSA/PBS.
?. [Cell proliferation]
Incubate with Alexa Fluor 594-conjugated secondary antibodies (1:1000 dilution) and HCS NuclearMask Blue stain for 1 hr at room temperature.
?. [Cell proliferation]
Wash cells five times in 80 mL/well PBS. On final wash, leave wells filled with 50 mL/well PBS.
?. [Cell proliferation]
Seal plates with foil seal and store at 4C before imaging.