TITLE: LENTIVIRAL TITRATION FOR HUMAN PLURIPOTENT STEM CELLS
AUTHORS: Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell

[DESCRIPTION]
We have developed a protocol for lentiviral titration of human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs). Concentrated lentiviral supernatants are added at various dilutions to adherent hPSCs in 48-well plates. Subsequent centrifugation, known as spinfection, ensures high efficiency in transduction. Transduction efficiency is quantified by determining the percentage of cells expressing Blue Fluorescent Protein (BFP) using Fluorescence Activated Cell Sorting (FACS).

[STEPS]
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
1. Coat 100 ul per well in a 48-well plate.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
2. Incubate the plate at room temperature for an hour and the plate is ready to be used.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
3. Seed 3x104 cells/cm2 in a 48 well plate with E8 flex media and RevitaCell after dissociating the cells with accutase.
SECTION: Day 0: Coating wells with VTN-N and seeding hPSCs
4. Incubate the cells overnight at 37ºC with 5% CO2 and 20.9% O2.
SECTION: Day 1: Titration of hPSCs with Lentiviral CRISPRi library supernatant
5. Prepare 15 ml tubes with E8 flex media and concentrated lentiviral supernatants in serial dilutions in the 48 well plate in the following manner. 

 

 

SECTION: Day 1: Titration of hPSCs with Lentiviral CRISPRi library supernatant
6. Aspirate the spent media with RevitaCell.
SECTION: Day 1: Titration of hPSCs with Lentiviral CRISPRi library supernatant
7. Add 200 ul /well for each viral dilution with the cells.
SECTION: Day 1: Titration of hPSCs with Lentiviral CRISPRi library supernatant
8. Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
SECTION: Day 2: Replace the media
9. Aspirate the viral supernatant media gently and immediately add maturation media.
SECTION: Day 2: Replace the media
10. Return the plate back to the incubator.
SECTION: Day 4: FACs Analysis
11. Aspirate the spent media.
SECTION: Day 4: FACs Analysis
12. Wash the cells 10 times with DPBS to remove the viral particles from the lentivirus transduced hPSCs.
 

SECTION: Day 4: FACs Analysis
13. Add 100 ul accutase and incubate the cells for 10 mins in the incubator.
SECTION: Day 4: FACs Analysis
14. Note: Ideally the hPSCs should dissociate as single cells.
SECTION: Day 4: FACs Analysis
15. Neutralize the accutase with E8 flex media and collect the cells into 1.5ml eppendorf tubes.
 

SECTION: Day 4: FACs Analysis
16. Centrifuge the cells at 300 g for 4 minutes.
SECTION: Day 4: FACs Analysis
17. Aspirate the spent media gently without disturbing the pellet. 
SECTION: Day 4: FACs Analysis
18. Resuspend the cells in 300 ul of FACs buffer.
SECTION: Day 4: FACs Analysis
19. Transfer the cells with the FACs buffer into FACs tubes.
SECTION: Day 4: FACs Analysis
20. Analyze the cells through flow cytometry to determine BFP positive cells.
21. The Multiplicity of Infection (MOI) for CRISPRi screen was quantified as the 0.1-0.1 or 10-30% of BFP-positive cells to ensure one gRNA enters one cell.
SECTION: Calculating the TU/ml
22. 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= NxFxVv

Where
T= Titer, (TU/mL)
N= Number of cells transduced
F= Fraction of cells with fluorescence
Vv= Virus volume
Detailed calculation 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
23. 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 x Vv)/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