TITLE: iNDI Papain Dissociation protocol for iNeurons Version 1
AUTHORS: Erika Lara Flores, Joel Reyes, Mark Cookson, Michael Ward

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparation of Reagents
1. Equilibrate EBSS/phenol red with 95%O2:5%CO2 in incubator for several hours of overnight.
SECTION: Preparation of Reagents
2. Reconstitute the Ovomucoid mixture (Papain inhibitor-PI) by adding 32 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 1 mL at 4 °C .

SECTION: Preparation of Reagents
3. Papain solution: Add 10 mL of TrypLE™ Express Enzyme (1X), no phenol red to the vial and place it in 37 °C  bath for 10 minutes, this vial has 10 units/ml.

 

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

SECTION: Preparation of Reagents
5. Trituration Medium:
30 mL of Neuronal Maturation medium (the same medium used for your differentiation)
30 µL of Rock inhibitor (1x)
1 vial of DNase I


SECTION: Preparation of Reagents
6. Papain Inhibitor media: 9 mL of Trituration medium + 1 mL of papain inhibitor.

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
7. Aspirate culture medium and wash gently with PBS (calcium/magnesium free).
SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
8. Aspirate PBS and wash 2 times with PBS-0.5mM EDTA
SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
9. Aspirate PBS-EDTA and add to one well of 6 well plate 1 mL  of Papain solution (10 units/ml) to cells, and incubate at 37 °C for 5 min .

 

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
10. 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.
SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
11. Add 1 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.

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
12. Collect cells into a sterile conical 15 mL tube and adjust volume to 5 mL with PBS or trituration medium.
SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
13. Centrifuge cells 5 min at 200 - 300 x g at Room temperature .

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
14. Remove supernatant and resuspend the cell pellet gently with 1 mL of Papain inhibitor medium to wash off the papain.

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
15. Centrifuge cells 5 min at 200 - 300 x g at Room temperature 

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
16. Aspirate supernatant and resuspend cell pellet in 1 mL  of Neuronal Maturation Medium.

SECTION: Dissociation protocol for iNeurons at day 28 of differentiation
17. After resuspending the cell pellet, count cells and use as necessary.