Mar 01, 2024
LENTIVIRAL TITRATION FOR HUMAN PLURIPOTENT STEM CELLS
This protocol is a draft, published without a DOI.
- Renuka Ravi Gupta1,2,3,4,
- Nona Farbehi1,2,3,5,
- hendersa 3,6,
- Vikram Khurana3,7,
- Gist Croft3,8,
- Lorenz Studer3,6,
- Joseph Powell1,2,3,4
- 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
Protocol Citation: Renuka Ravi Gupta, Nona Farbehi, hendersa , Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell 2024. LENTIVIRAL TITRATION FOR HUMAN PLURIPOTENT STEM CELLS. protocols.io https://protocols.io/view/lentiviral-titration-for-human-pluripotent-stem-ce-c9wnz7de
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, lentiviral titration for human pluripotent stem cell, human pluripotent stem cell, induced pluripotent stem cell, pluripotent stem cell, protocol for lentiviral titration, lentiviral titration, using fluorescence activated cell sorting, fluorescence activated cell sorting, concentrated lentiviral supernatant, human embryonic stem cell, embryonic stem cell, expressing blue fluorescent protein, blue fluorescent protein, activated cell, subsequent centrifugation
Funders Acknowledgements:
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).
Attachments
Materials
| A | B | C | |
| MATERIAL | COMPANY | CATALOG | |
| 48 well TC treated plate | Falcon | 353078 | |
| 15ml polypropylene centrifuge tubes | Falcon | 352096 | |
| 5ml serological pipettes | Corning | 4487 | |
| 10ml serological pipettes | Corning | 4488 | |
| DNA Low-bind tubes 1.5ml | Eppendorf | 022431021 | |
| P1000 tip | Neptune | BT1250 | |
| FBS | Bovogen | 2008A | |
| Dulbecco Phosphate Buffer Saline (DPBS) | Thermo Fisher Scientific | 14190-250 | |
| E8 Flex media Kit | Thermo Fisher Scientific | A2858501 | |
| RevitaCell Supplement(100x) | Thermo Fisher Scientific | A2644501 | |
| Accutase | StemCell Technologies | 7922 | |
| Vitronectin-N (VTN-N) | Thermo Fisher Scientific | A14700 |
REAGENT
| A | B | |
| FACS Buffer (PBS +2% FBS) | ||
| REAGENT | VOLUME IN mL | |
| PBS | 49 | |
| FBS | 1 | |
Troubleshooting
Day 0: Coating wells with VTN-N and seeding hPSCs
Coat 100 ul per well in a 48-well plate.
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
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
Aspirate the viral supernatant media gently and immediately add maturation media.
Return the plate back to the incubator.
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
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/
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 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:
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