Aug 08, 2023

Public workspaceiNDI Papain Dissociation protocol for iNeurons Version 1

DOI
dx.doi.org/10.17504/protocols.io.kxygx392dg8j/v1
  • Erika Lara Flores1,
  • Joel Reyes2,
  • Mark Cookson1,
  • Michael Ward1
  • 1National Institutes of Health, NIH Center for Alzheimer's and Related Dementias (CARD);
  • 2National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS)
Erika Lara Flores
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DOI: dx.doi.org/10.17504/protocols.io.kxygx392dg8j/v1
Protocol Citation: Erika Lara Flores, Joel Reyes, Mark Cookson, Michael Ward 2023. iNDI Papain Dissociation protocol for iNeurons Version 1. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx392dg8j/v1
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: August 08, 2023
Last Modified: August 08, 2023
Protocol Integer ID: 86180
Abstract
Proteolytic enzymes are widely used in cell dissociation and papain has proved less damaging with some tissues and neuronal cultures and more effective than other proteases. Here we describe a papain dissociation protocol that has been used on our iPSC-derived neurons for scRNAseq and FACS assays redouts.
Materials
ReagentPapain Dissociation SystemWorthington Biochemical CorporationCatalog #LK003153

ReagentTrypLE™ Express Enzyme (1X) no phenol redThermo Fisher ScientificCatalog #12604013

PBS

PBS-EDTA 0.05mM

PBS-0.04% BSA (For scRNAseq cell preparation, if applicable)


Preparation of Reagents
Preparation of Reagents
Equilibrate EBSS/phenol red with 95%O2:5%CO2 in incubator for several hours of overnight.
Reconstitute the Ovomucoid mixture (Papain inhibitor-PI) by adding Amount32 mL of EBSS and allow contents to dissolve. This will yield solution at an effective concentration of 10mg of ovomucoid inhibitor and 10mg of albumin per mL. Reconstitute for the first use, then store Amount1 mL at Temperature4 °C .

Papain solution: Add Amount10 mL of TrypLE™ Express Enzyme (1X), no phenol red to the vial and place it in Temperature37 °C bath for 10 minutes, this vial has 10 units/ml.

Note
When working with i3Neurons (WTC11 line), dissolve the papain with 20 mL of TrypLE™ Express Enzyme (1X).

DNase I vial D2: Add 500ul of EBSS media and mix gently (2000 units/ml).

Trituration Medium:
Amount30 mL of Neuronal Maturation medium (the same medium used for your differentiation)
Amount30 µL of Rock inhibitor (1x)
1 vial of DNase I


Papain Inhibitor media: Amount9 mL of Trituration medium + Amount1 mL of papain inhibitor.

Dissociation protocol for iNeurons at day 28 of differentiation
Dissociation protocol for iNeurons at day 28 of differentiation
5m
5m
Aspirate culture medium and wash gently with PBS (calcium/magnesium free).
Aspirate PBS and wash 2 times with PBS-0.5mM EDTA
Aspirate PBS-EDTA and add to one well of 6 well plate Amount1 mL of Papain solution (10 units/ml) to cells, and incubate at Temperature37 °C for Duration00:05:00 .

Note
Incubation time can vary and go up to 30 minutes and it depends on cell line, confluency and maturation day.
Incubation times for i3Neurons (WTC11):
Day 7: 3 minutes
Day 10: 5 minutes
Day 14: 10 minutes

5m
Aspirate papain solution when the cell bodies have an halo morphology like for and EDTA split. If the incubation is longer (up to 30 minutes) the cells will detach into papain solution, then continue with the next step.
Add Amount1 mL of trituration medium per well of a 6-well plate to dissociate cells and pipette medium over the plate up and down to detach cells and dissociate them as single-cell suspension.

Collect cells into a sterile conical 15 mL tube and adjust volume to 5 mL with PBS or trituration medium.
Centrifuge cells Duration00:05:00 at 200 - 300 x g at TemperatureRoom temperature .

5m
Remove supernatant and resuspend the cell pellet gently with Amount1 mL of Papain inhibitor medium to wash off the papain.

Centrifuge cells Duration00:05:00 at 200 - 300 x g at TemperatureRoom temperature

5m
Aspirate supernatant and resuspend cell pellet in Amount1 mL of Neuronal Maturation Medium.

After resuspending the cell pellet, count cells and use as necessary.

Note
For scRNAseq 10X genomics assay resuspend in cold PBS-0.04%BSA.
For FACS assay resuspend in cold PBS-0.5mM EDTA.