006005004104.GOT
Date: 21-FEB-2023
Author: JH. Version: 5

VIABILITY STAINING PROTOCOL

Calcein AM and PropidiumIodide
This is a suggested procedure, please adjust according to your experimental needs.

Protocol aim

Materials needed

The aim of this protocol is to provide instructions for
performing live and dead staining using Calcein AM
and propidium iodide (PI) for imaging of live
respectively dead cells in 3D constructs of all
CELLINKs bioinks.

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Calcein AM (Invitrogen™ eBioscience™,product number 15560597)
Propidium iodide (Sigma-Aldrich, productnumber 81845-25MG)
Cell-laden bioprinted constructs
Hank’s Balanced Salt Solution (HBSS+/+)
5 mL amber Eppendorf tubes
Inverted fluorescence microscope with filter
sets for FITC and Texas Red

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KEEP STOCKS, WORKING SOLUTIONS AND STAINED CELLS AWAY FROM LIGHT SOURCES.

ProtocolThis protocol is optimized for CELLINKs bioinks, printed as droplets in 96-well plates. For larger structures, pleaseadjust volumes and incubation times. Larger constructs need larger staining solution volumes to fully cover theconstruct and longer incubation times for the dye to penetrate to the center of the construct.

1.

Preparing stock solutions

MATERIAL
Calcein AMDMSOPIHBSS+/+DESCRIPTION•

If stock solutions already are prepared, move to Step 2.
••

2.

To prepare 1 mM of Calcein AM stock solution: add 50.3 µL DMSO into a vial of Calcein AMand mix gently. Aliquot and store stock solutions at -20°C.
To prepare 1 mg/mL of PI stock solution: weigh 1 mg of PI in an Eppendorf tube. Add 1 mL ofHBSS+/+ to the tube with PI and mix properly. Make sure that all PI powder isdissolved. Aliquot and store stock PI solution at 4°C.

Pre-washing

MATERIAL
HBSS+/+DESCRIPTION••

3.

Transfer the constructs (triplicates of each condition is preferred) to a separate well plate with wells filledwith HBSS+/+, wash for 5 min at 37°C.
Optional: For a dead control, remove HBSS+/+ and add ethanol to cover the construct. Remove after 5min and add HBSS+/+.

Calcein AM working solution

MATERIAL
Stock solution of Calcein AM
4 mL HBSS+/+
5 mL amber Eppendorf tube

DESCRIPTION••Thaw stock solution of Calcein AM.
Add 1 µL Calcein AM stock solution into the Eppendorf tube with 4 mL of HBSS+/+ to make the Calcein
AM working solution.
Note: Staining solution should be used within 2 hours from preparation.
Note: Alternatively dilute Calcein AM in medium instead of HBSS+/+.

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•Remove the HBSS+/+ from constructs and add 100 µL Calcein AM working solution to each well,incubate at 37°C for 20 min.

Note: The amount of staining solution required per construct depends on sample size. Add enough stainingsolution to cover the construct.

4.

PI working solutionMATERIAL
Stock solution of PI
4 mL HBSS+/+
5 mL amber Eppendorf tube

DESCRIPTION•Add 400 µL of PI stock solution into an amber tube with 4 mL HBSS+/+ to make the PI working solution.
Note: Staining solution should be used within 2 hours from preparation.
•Remove Calcein AM working solution and add 100 µL PI staining solution, incubate constructs for 6 min
at 37°C.
Note: Required amount of staining solution needed depends on sample. Add enough staining solution tocover construct.

5.

WashingMATERIAL
HBSS+/+DESCRIPTION••Remove PI working solution and wash twice for 15 min in HBSS+/+ at 37°C.
Store in HBSS+/+ until imaging.

Note: Wash by adding and removing HBSS+/+ once.

6.

Imaging

MATERIAL
Fluorescent microscope
Image analysis software

DESCRIPTION•••

Bring cell-laden constructs to fluorescent microscope for imaging.
Capture images in the green (FITC/488) and red (TexasRed/570) channels as Z-stacks.
Assess the viability percentage using a software for image analysis, e.g. Image J to count the number oflive and dead cells per image.

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