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: 95918
Keywords: ASAPCRN, Lentiviral Titration, Human Pluripotent Stem Cells, CRISPRi
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000472
Abstract
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).
Incubate the plate at room temperature for an hour and the plate is ready to be used.
Seed 3x104 cells/cm2 in a 48 well plate with E8 flex media and RevitaCell after dissociating the cells with accutase.
Incubate the cells overnight at 37ºC with 5% CO2 and 20.9% O2.
Day 1: Titration of hPSCs with Lentiviral CRISPRi library supernatant
Day 1: Titration of hPSCs with Lentiviral CRISPRi library supernatant
Prepare 15 ml tubes with E8 flex media and 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.
A
B
C
D
E
F
G
H
I
J
K
DILUTION
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Media(ul)
600
600
600
600
600
600
600
600
600
600
Viral supernatant(ul)
600
600 of 1/2
600 of 1/4
600 of 1/8
600 of 1/16
600 of 1/32
600 of 1/64
600 of 1/128
600 of 1/256
600 of 1/512
Table: 1 Serial dilution of concentrated lentiviral supernatant to determine lentiviral titer in TU/mL
Aspirate the spent media with RevitaCell.
Add 200 ul /well for each viral dilution with the cells.
Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
Day 2: Replace the media
Day 2: Replace the 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 lentivirus transduced hPSCs.
Note
Be very gentle while doing the washes as the cells tend to lift off during the wash step.
Add 100 ul accutase and incubate the cells for 10 mins in the incubator.
Note: Ideally the hPSCs should dissociate as single cells.
Neutralize the accutase with E8 flex media and collect the cells into 1.5ml eppendorf tubes.
Note
Use a P1000 tip to pipette the cells up and down to break them into single cell suspension.
Centrifuge the cells at 300 g for 4 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 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.
Calculating the TU/ml
Calculating the 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= NxFxVv
Where
T= Titer, (TU/mL)
N= Number of cells transduced
F= Fraction of cells with fluorescence
Vv= Virus volume