iNDI Papain Dissociation protocol for iNeurons Version 1
Erika Lara Flores, Mark Cookson, Michael Ward, Joel Reyes
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.
Steps
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 32mL 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 1mL at 4°C .
Papain solution: Add 10mL 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.
DNase I vial D2: Add 500ul of EBSS media and mix gently (2000 units/ml).
Trituration Medium:
30mL of Neuronal Maturation medium (the same medium used for your differentiation)
30µL of Rock inhibitor (1x)
1 vial of DNase I
Papain Inhibitor media: 9mL of Trituration medium + 1mL of papain inhibitor.
Dissociation protocol for iNeurons at day 28 of differentiation
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 1mL of Papain solution (10 units/ml) to cells, and incubate at 37°C for 0h 5m 0s .
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 1mL 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 0h 5m 0s at 200 - 300 x g at Room temperature .
Remove supernatant and resuspend the cell pellet gently with 1mL of Papain inhibitor medium to wash off the papain.
Centrifuge cells 0h 5m 0s at 200 - 300 x g at Room temperature
Aspirate supernatant and resuspend cell pellet in 1mL of Neuronal Maturation Medium.
After resuspending the cell pellet, count cells and use as necessary.
