Mar 01, 2024

Public workspaceLENTIVIRAL TITRATION FOR EARLY POST- MITOTIC DOPAMINERGIC NEURONS

This protocol is a draft, published without a DOI.
  • 1Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 2Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
  • 4School of Medical Science, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 5Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 6The Centre for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA;
  • 7Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
  • 8The New York Stem Cell Foundation Research Institute, New York, NY, USA
Renuka Ravi Gupta
Open access
Protocol Citation: Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell 2024. LENTIVIRAL TITRATION FOR EARLY POST- MITOTIC DOPAMINERGIC NEURONS. protocols.io https://protocols.io/view/lentiviral-titration-for-early-post-mitotic-dopami-c9wvz7e6
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: In development
We are still developing and optimizing this protocol
Created: February 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95925
Keywords: ASAPCRN, Lentiviral Production, CRISPRi, dopaminergic neuron differentiation, Perturb-Seq
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000472
Abstract
iPSCs- derived neurons are particularly challenging cells for genetic screening. Hence we develop a protocol for lentiviral titration of mDA neurons where mDA neuronal cell suspension combined with concentrated lentiviral supernatant are added at different dilutions in 48 well plates. Subsequent centrifugation (spinfection) was performed to achieve high efficiency transduction. The transduction efficiency is determined as a percentage of BFP- positive cells through FACS (Fluorescence Activated Cell Sorting).
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LENTIVIRAL TITRATION FOR EARLY POST- MITOTIC DOPAMINERGIC NEURONS.docx
LENTIVIRAL TITRATION...
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Materials

ABC
MATERIALCOMPANYCATALOG
48 well TC treated plateFalcon353078
15ml polypropylene centrifuge tubesFalcon352096
5ml serological pipettesCorning4487
10ml serological pipettesCorning4488
DNA Low-bind tubes 1.5mlEppendorf022431021
P1000 tipNeptuneBT1250
FBSBovogen2008A
DPBSThermoFisher Scientific14040133
Hank’s Balanced Salt Solution (HBSS)ThermoFisher Scientific14175-095
Neuronal Isolation Neuronal Enzyme (with Papain)ThermoFisher Scientific88285
Neurobasal MediaThermoFisher Scientific21103049
B27 w/o vit AThermoFisher Scientific12587-010
L-glutamineThermoFisher ScientificL3000015
Pen-StrepThermoFisher Scientific12260
BDNF (Brain Derived Neurotrophic Factor)R&D248-BDB
GDNF (Glial Cell line Derived Neurotrophic Factor)Peprotech450-10
Ascorbic AcidSigma4034
cAMPSigmaD0627
TGF-B (Transforming Growth Factor - b)R&D243-B3
DAPT Tocris2634
Polyornithine (PO)SigmaP3655
Cultrex Mouse Laminin IR&D3400-010-1
FibronectinCorningFAL356008
REAGENT COMPOSITION
AB
MEDIA 2
REAGENTVOLUME IN ML
Neurobasal Media480
B27 without Vit A (10x)10
Pen-Strep5
L-Glutamine5

ABCD
MATURATION MEDIA (MM)
REAGENTSTOCK SOLUTIONWORKING SOLUTIONVOLUME IN ul
Media 2 --24796
BDNF10 ug/ml20ng/ml50
GDNF10 ug/ml20 ng/ml50
AA100mM200uM50
cAMP100mM200uM50
DAPT100mM10uM2.5
TGF-B20 ug/ml1ng/ml1.25

AB
FACS BUFFER (PBS +2% FBS)
REAGENTVOLUME IN mL
PBS49
FBS1

Before start
hESC CRISPRi dCAS9 are differentiated to D25 according to the following protocol:
CITATION
Tae Wan Kim, Jinghua Piao, So Yeon Koo, Sonja Kriks, Sun Young Chung, Doron Betel, Nicholas D. Socci, Se Joon Choi, Susan Zabierowski, Brittany N. Dubose, Ellen J. Hill, Eugene V. Mosharov, Stefan Irion, Mark J. Tomishima, Viviane Tabar, Lorenz Studer. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. protocols.io.
LINK
https://protocols.io/view/biphasic-activation-of-wnt-signaling-facilitates-t-bu7znzp6
At D25, the cells were sorted by MACs sorting to obtain pure population dopaminergic mDA neurons.

CITATION
Tae Wan Kim. Dopamine neuron enrichment using MACS. protocols.io.
LINK
https://protocols.io/view/dopamine-neuron-enrichment-using-macs-cyrfxv3n

Day -1: Coating wells with Poly - L ornithine(PO)
Day -1: Coating wells with Poly - L ornithine(PO)
Coat 500 ul per well in a 48-well plate with 15 ug/ml PO in DPBS.
Incubate the plate overnight at 37ºC with 5% CO2 and 20.9% O2.
Day 0: Coating wells with Laminin and Fibronectin
Day 0: Coating wells with Laminin and Fibronectin
Thaw Fibronectin and Laminin on ice.
Aspirate 250ul of coated PO from each well of the 48 well plate and wash the wells with 1 ml of DPBS. Repeat two more times for a total of 3 x DPBS washes.
Note
Do not let the wells dry out.

Aspirate DPBS and add 500 ul of 2ug/ml Fibronectin and 1ug/ml Laminin in cold DPBS.
Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
Thaw the viral stock on ice.
Prepare 15 ml tubes with 200000 D25 pure population mDA neuronal suspension (CD49 neg) with concentrated lentiviral supernatants in serial dilutions in the 48 well plate in the following manner.
Note
Make sure to mix well by gentle pipetting. Change tips after making up each dilution. Titration was done in triplicates.

ABCDEFGHIJK
DILUTION
1/21/41/81/161/321/641/1281/2561/5121/1024
Cell+MM media600ul600ul600ul600ul600ul600ul600ul600ul600ul600ul
Viral supernatant600ul600ul of 1/2600ul of 1/4600ul of 1/8600ul of 1/16600ul of 1/32600ul of 1/64600ul of 1/128600ul of 1/256600ul of 1/512
Table: 1 Serial dilution of concentrated lentiviral supernatant to determine lentiviral titer in TU/mL

Aspirate the fibronectin/laminin coating and proceed immediately to the next step.
Add 200 ul/well for each viral dilution with the cells.
To increase the transduction efficiency, centrifuge the plate at 300g for 20 minutes at 25 C.
Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
Day 2: Replace media
Day 2: Replace media
Aspirate the viral supernatant media gently and immediately add maturation media.
Return the plate back to the incubator.
Day 4: FACs Analysis
Day 4: FACs Analysis
Aspirate the spent media.
Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons.
Note: The neurons are sturdy and do not lift off during the washes. However look under the microscope during the washes to avoid the neurons lifting off.
Add 100ul HBSS +papain and incubate the neurons for 45 mins in the incubator.
Note
Ideally the neurons should dissociate as single cells.

Neutralize the papain with maturation media and collect the cells into 1.5ml eppendorf tubes.
Note
If the neurons are still present as a sheet or have clumps, use a P1000 tip, pipette the cells up and down to break them into single cell suspension.

Centrifuge the cells at 300 g for 5 minutes.
Aspirate the spent media gently without disturbing the pellet.
Resuspend the cells in 300 ul of FACs buffer.
Transfer the cells with the FACs buffer into FACs tubes.
Analyze the cells through flow cytometry to determine BFP positive cells.
The MOI for CRISPRi screen was quantified as the 10%-30% of BFP-positive cells to ensure one gRNA enters one cell.
Calculating the Lentiviral Titer in TU/ml
Calculating the Lentiviral Titer in TU/ml
Method 1: Calculating using dilution Factor
T= (NXFXD)/Vt Where T= Titer, (TU/mL) N=Number of cells transduced F= Fraction of cells with fluorescence D=Dilution Factor Vt=Transduction volume in mL

Method 2: Calculating using volume of virus
T= (NxF)/Vv Where, T= Titer, (TU/mL) N=Number of cells transduced F= Fraction of cells with fluorescence Vv=Virus volume

Detailed protocol for lentiviral titration for the virus can be found in the following link: https://www.addgene.org/protocols/fluorescence-titering-assay/
Calculating Virus volume for required MOI
Calculating Virus volume for required MOI
For Perturb seq, to restrict the viral integration in such a way that one virus infects one cell, we keep the MOI between 0.1-0.3.
Calculating the virus volume, for MOI (0.1-0.3).
MOI=(T xVv)/N

Where, T= Titer, (TU/mL) N=Number of cells transduced Vv=Virus volume

Detailed protocol for calculating MOI can be found in the following link:
https://info.abmgood.com/multiplicity-of-infection-moi

Protocol references
Calculation for lentiviral titration for the virus can be found in the following link: https://www.addgene.org/protocols/fluorescence-titering-assay/

Calculation of MOI can be found in the following link: https://info.abmgood.com/multiplicity-of-infection-moi





Citations
Tae Wan Kim, Jinghua Piao, So Yeon Koo, Sonja Kriks, Sun Young Chung, Doron Betel, Nicholas D. Socci, Se Joon Choi, Susan Zabierowski, Brittany N. Dubose, Ellen J. Hill, Eugene V. Mosharov, Stefan Irion, Mark J. Tomishima, Viviane Tabar, Lorenz Studer. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use
https://protocols.io/view/biphasic-activation-of-wnt-signaling-facilitates-t-bu7znzp6
Tae Wan Kim. Dopamine neuron enrichment using MACS
https://protocols.io/view/dopamine-neuron-enrichment-using-macs-cyrfxv3n