TITLE: LENTIVIRAL TITRATION FOR EARLY POST- MITOTIC DOPAMINERGIC NEURONS
AUTHORS: Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell

[DESCRIPTION]
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).

[BEFORE_START]
hESC CRISPRi dCAS9 are differentiated to D25 according to the following protocol: 
 
At D25, the cells were sorted by MACs sorting to obtain pure population dopaminergic mDA neurons.

 


[STEPS]
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
1. Coat 500 ul per well in a 48-well plate with 15 ug/ml PO in DPBS.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
2. Incubate the plate overnight at 37ºC with 5% CO2 and 20.9% O2.
SECTION: Day 0: Coating wells with Laminin and Fibronectin
3. Thaw Fibronectin and Laminin on ice.
SECTION: Day 0: Coating wells with Laminin and Fibronectin
4. 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.
 

SECTION: Day 0: Coating wells with Laminin and Fibronectin
5. Aspirate DPBS and add 500 ul of 2ug/ml Fibronectin and 1ug/ml Laminin in cold DPBS.
SECTION: Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
6. Thaw the viral stock on ice.
SECTION: Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
7. 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.
 

 

SECTION: Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
8. Aspirate the fibronectin/laminin coating and proceed immediately to the next step.
SECTION: Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
9. Add 200 ul/well for each viral dilution with the cells.
SECTION: Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
10. To increase the transduction efficiency, centrifuge the plate at 300g for 20 minutes at 25 C.
SECTION: Day 1: Titration of D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
11. Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
SECTION: Day 2: Replace media
12. Aspirate the viral supernatant media gently and immediately add maturation media.
SECTION: Day 2: Replace media
13. Return the plate back to the incubator.
SECTION: Day 4: FACs Analysis
14. Aspirate the spent media.
SECTION: Day 4: FACs Analysis
15. Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons.
SECTION: Day 4: FACs Analysis
16. 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.
SECTION: Day 4: FACs Analysis
17. Add 100ul HBSS +papain and incubate the neurons for 45 mins in the incubator.
 

SECTION: Day 4: FACs Analysis
18. Neutralize the papain with maturation media and collect the cells into 1.5ml eppendorf tubes.
 

SECTION: Day 4: FACs Analysis
19. Centrifuge the cells at 300 g for 5 minutes.
SECTION: Day 4: FACs Analysis
20. Aspirate the spent media gently without disturbing the pellet.
SECTION: Day 4: FACs Analysis
21. Resuspend the cells in 300 ul of FACs buffer.
SECTION: Day 4: FACs Analysis
22. Transfer the cells with the FACs buffer into FACs tubes.
SECTION: Day 4: FACs Analysis
23. Analyze the cells through flow cytometry to determine BFP positive cells.
SECTION: Day 4: FACs Analysis
24. The MOI for CRISPRi screen was quantified as the 10%-30% of BFP-positive cells to ensure one gRNA enters one cell.
SECTION: Calculating the Lentiviral Titer in TU/ml
25. 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/
SECTION: Calculating Virus volume for required MOI
26. 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.
SECTION: Calculating Virus volume for required MOI
27. 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