TITLE: Single-gene short-term CRISPR ko viability assay
AUTHORS: Benjamin Gaeta, Tsukasa Shibue, Brenton Paolella, Francisca Vazquez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">The Cancer Dependency Map Project </span><span>is using genome-scale CRISPR loss-of-function screens on a large number of  cancer models to systematically identify selective genetic dependencies, which in turn represent potential therapeutic targets. As we identify potential targets through this project, it is becoming increasingly important to have a robust validation strategy to de-risk individual targets before embarking on costly therapeutic projects. </span></div><div class = "text-block"><span>Here we describe an experimental strategy for assessing the</span><span style = "font-weight:bold;"> short-term viability effect </span><span>after knocking out a gene of interest via CRISPR/Cas9 using a commercially-available luminescent cell viability assay reagent (Cell-titer Glo [Promega]) in a 96-well format.  The robustness and reproducibility of this assay are critically dependent on two experimental parameters: seeding density of the cells and the lentivirus infection efficiency. We provide standardized protocols for the rigorous determination of both of these parameters. </span></div><div class = "text-block">In addition, given that CRISPR-mediated gene knockout penetrance differ across cell lines, the use of standardized controls is necessary for the comparative assessment of the strength of the dependency across cell lines. Moreover, there is a non-specific toxicity effect resulting from the DNA double-strand breaks caused by the CRISPR/Cas9 system. It is therefore important to use sgRNAs targeting intragenic regions, which serve as negative controls to account for this non-specific toxicity. We have tested and identified the sequences of negative and positive sgRNAs controls (pan-essential genes) that can be used to scale the cell viability read-outs. </div><div class = "text-block">Finally, we also provide a series of templates to record and normalize the data produced throughout this assay. </div></div>

[STEPS]
?. [Assay Development]
(Day 0) Template/Planning: create a copy of the "Viability Assay Optimization" template and fill out the information.
?. [Assay Development]
(Day 0) Make Viral Master Plate: prepare (a) master plate(s) of BRD003 lenti-virus that contains the following volume of virus per 40 uL of total volume  ABCDEFGHIJKLM11234567891011122A            3B 01.251.251.251.251.251.251.251.251.25 4C 02.52.52.52.52.52.52.52.52.5 5D 0555555555 6E 0101010101010101010 7F 0202020202020202020 8G 0404040404040404040 9H            * NOTE- up to 4 cell types/one master plate; if seeding > 4 cell types, make multiple master plates
ABCDEFGHIJKLM11234567891011122A            3B 01.251.251.251.251.251.251.251.251.25 4C 02.52.52.52.52.52.52.52.52.5 5D 0555555555 6E 0101010101010101010 7F 0202020202020202020 8G 0404040404040404040 9H
?. [Assay Development]
Prepare (# of cell types) x 400 uL of BRD003 virus
?. [Assay Development]
Fill column 2, rows B-G and columns 3-11, rows B-F (wells w. black numbers) with (# of cell types) x 50 uL of R10 medium
?. [Assay Development]
Aliquot (# of cell types) x 100 uL of BRD003 virus into columns 3-11, row G (wells w. red numbers)
?. [Assay Development]
Transfer (# of cell types) x 50 uL of virus-containing medium from columns 3-11, row G to columns 3-11, row F and mix well (not well itself but its content); change chips of multichannel pipetter
?. [Assay Development]
Transfer (# of cell types) x 50 uL of virus-containing medium from columns 3-11, row F to columns 3-11, row E and mix well (not well itself but its content); change chips of multichannel pipetter
?. [Assay Development]
Transfer (# of cell types) x 50 uL of virus-containing medium from columns 3-11, row C to columns 3-11, row B and mix well (not well itself but its content)
?. [Assay Development]
(Day 0) Cell Seeding: seed the numbers of cells in 100 uL medium (containing 5.6 ug/mL polybrebne [H9268, SIgma-Aldrich]) indicated in this table in two 96 well plates (per cell line):  ABCDEFGHIJKLM1(Plate  I)1234567891011122A            3B 1000100010001000100010001000200020002000 4C 1000100010001000100010001000200020002000 5D 1000100010001000100010001000200020002000 6E 1000100010001000100010001000200020002000 7F 1000100010001000100010001000200020002000 8G 1000100010001000100010001000200020002000 9H              ABCDEFGHIJKLM1(Plate II)1234567891011122A            3B 2000200020002000400040004000400040004000 4C 2000200020002000400040004000400040004000 5D 2000200020002000400040004000400040004000 6E 4000200020002000400040004000400040004000 7F 4000200020002000400040004000400040004000 8G 4000200020002000400040004000400040004000 9H
ABCDEFGHIJKLM1(Plate  I)1234567891011122A            3B 1000100010001000100010001000200020002000 4C 1000100010001000100010001000200020002000 5D 1000100010001000100010001000200020002000 6E 1000100010001000100010001000200020002000 7F 1000100010001000100010001000200020002000 8G 1000100010001000100010001000200020002000 9H
ABCDEFGHIJKLM1(Plate II)1234567891011122A            3B 2000200020002000400040004000400040004000 4C 2000200020002000400040004000400040004000 5D 2000200020002000400040004000400040004000 6E 4000200020002000400040004000400040004000 7F 4000200020002000400040004000400040004000 8G 4000200020002000400040004000400040004000 9H
?. [Assay Development]
make 6 x (# of cell type) mL medium containing 5.6 ug/mL polybrene (this is ‘A’).
?. [Assay Development]
make a suspension of 40K cell/10 mL medium (w/ 5.6 ug/mL polybrene) (this is ‘B’).
?. [Assay Development]
Aliquot 75 uL of A into Plate I, columns 2-8 .
?. [Assay Development]
Aliquot 50 uL of A into Plate I, columns 9-11; Plate II, columns 2-5 (except for 2E, 2F, 2G).
?. [Assay Development]
Aliquot 25 uL  of B into Plate I, columns 2-8.
?. [Assay Development]
Aliquot 50 uL of B into Plate I, columns 9-11; Plate II, columns 2-5 (except for 2E, 2F, 2G).
?. [Assay Development]
Aliquot 100 uL  of B into Plate II, columns 6-11 and 2E, 2F, 2G.
?. [Assay Development]
(Day 0) Infection: transfer 40 uL of (virus-containing) medium from the virus master plate to the corresponding well of cell-seeded plate; centrifuge at 900 x g, 1.5h at 37°C.
?. [Assay Development]
(Day 1) Medium change/Puro selection: change medium with 200 uL of fresh medium
- Puro (+): For only Plate 1, columns 6-8 and Plate II, columns 3-5, 9-11, use 2 ug/mL Puromycin-containing medium.
?. [Assay Development]
(Day 3) change medium (Puro (-) or (+)).
?. [Assay Development]
(Day 5) change medium (Puro (-) or (+)).
?. [Assay Development]
(Day 7) CTG measurement: Perform the CellTiter-Glo protocol provided by Promega and paste the CTG values for each cell line under the "Assay Dev" tab of the "Viability Assay Optimization" template.
?. [Determining optimal seeding density ]
For each of the seeding densities – i.e., 1000, 2000, 4000 cells/well --, determine the minimum volume of virus that yields (mean CTG value of Puro (+) wells) > 0.8 x (mean CTG value of Puro (-) wells): Set the mean Puro (-) CTG value for this volume of virus as C1000 (or C2000, C4000); check if the C value determined here is larger than ‘0.7 x (corresponding mean CTG value of infection/Puro (-) sample)’.
?. [Determining optimal seeding density ]
If C4000≥ 1.8 x C2000, use 4000 cells/well (if not, go to 11).
?. [Determining optimal seeding density ]
If 1.5 x C2000 ≤ C4000 2000, use  3000 cell//well (if not, go to 12).
?. [Determining optimal seeding density ]
If C2000≥ 1.8 x C1000, use 2000 cells/well (if not, go to 13).
?. [Determining relative infection sensitivity]
Determine the minimum volume of virus that gives (mean CTG value of Puro (+) wells) > 0.8 x (mean CTG value of Puro (-) wells) for the detemined seeding density of corresponding cells. If an interpolated number of cells is used (3000 or 1500 cells/ well), take the mean virus volume for the "4000" and "2000" values. Based on this virus volume (which reflects sensitivity to viral infection/viral toxicity), categorize cell lines used for a single project (usually around 10 cell lines of varying lineages) into 3 classes: HIGH/MEDIUM/LOW infection sensitivity. When you prepare a set of virus for viability assay, pick one cell line from each class and titrate all the virus against these cell lines (see an example below):  AB1sensitivityExample cell lines2LOWSNU503_colon, JIMT1_breast, HCC95_lung, KYSE410_esophagus3MEDSW837_colon, HCC15_lung, KYSE450_esophagus4HIGHHCC202_breast, AU565_breast, SKBR3_breast, ZR751_breast
AB1sensitivityExample cell lines2LOWSNU503_colon, JIMT1_breast, HCC95_lung, KYSE410_esophagus3MEDSW837_colon, HCC15_lung, KYSE450_esophagus4HIGHHCC202_breast, AU565_breast, SKBR3_breast, ZR751_breast
?. [Titration of Virus for Viability Assay]
Measure the titers of 9 types of virus prepared/aliquoted as below, using 3 representative cells lines -- 1 from HIGH, MEDIUM, and LOW infection sensitivity (as identified by the Week1 'Assay Dev' [above].
?. [Titration of Virus for Viability Assay]
Prepare virus for each of the:3 negative controls (BRD003-sgLacZ/-sgCH2-2/-sgAAVS1)3 positive controls (BRD003-sgPOLR2D/sgSF3B1/-sgKIF11)3 target-specific guides (BRD-003-sgXXX-A/-sgXXX-B/-sgXXX-C) Prepare virus in 1x15cm dish; this will yield approx 17 mL of virus. Aliquot the virus into 1.5 mLx11 vials and assemble as below:  ABCDEFGHIJ1sgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZ2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-23sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS14sgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2D5sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B16sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF117sgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-A8sgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-B9sgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-C10sgLacZsgCH2-2sgAAVS1sgPOLR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C---sgRNA sequence for all controls:  AB1sgLacZ (non-targeting control)AACGGCGGATTGACCGTAAT2sgCh2-2 (intergenic control)GGTGTGCGTATGAAGCAGTG3sgAAVS1 (intergenic control)GGGCCACTAGGGACAGGAT4sgPOLR2D (positive control)AGAGACTGCTGAGGAGTCCA5sgSF3B1 (positive control)AAAAGGATCAAGACGCTCTG6sgKIF11 (positive control)TCTTGTGTAGGAGTATACGG
ABCDEFGHIJ1sgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZsgLacZ2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-2sgCH2-23sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS1sgAAVS14sgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2DsgPOLR2D5sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B1sgSF3B16sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF11sgKIF117sgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-AsgTarget-A8sgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-BsgTarget-B9sgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-CsgTarget-C10sgLacZsgCH2-2sgAAVS1sgPOLR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C---
AB1sgLacZ (non-targeting control)AACGGCGGATTGACCGTAAT2sgCh2-2 (intergenic control)GGTGTGCGTATGAAGCAGTG3sgAAVS1 (intergenic control)GGGCCACTAGGGACAGGAT4sgPOLR2D (positive control)AGAGACTGCTGAGGAGTCCA5sgSF3B1 (positive control)AAAAGGATCAAGACGCTCTG6sgKIF11 (positive control)TCTTGTGTAGGAGTATACGG
?. [Titration of Virus for Viability Assay]
Pick 3 cell lines representing HIGH/MEDIUM/LOW infection sensitivity based on the ‘Assay Development’ resulte.g. SNU503(LOW), SW837 (MEDIUM), HCC202(HIGH)
?. [Titration of Virus for Viability Assay]
(Day 1) Change medium (Plate A: just medium; Plate B: medium with 2 uG/uL Puro)
?. [Titration of Virus for Viability Assay]
(Day 4) Read CTG
?. [Titration of Virus for Viability Assay]
Determine appropriate amount of virus to add per cell line
?. [Titration of Virus for Viability Assay]
Plot the titration result (CTG value; see below)
?. [Titration of Virus for Viability Assay]
Determine the amount of virus (X) that fulfills both:A) CTG ​virus=x, Puro(-) / CTG​ virus=0, Puro(-) ≥​0.8​, B) ​CTG​ virus=x, Puro (+) / CTG​ virus=0, Puro(-) ≥​0.8
?. [Titration of Virus for Viability Assay]
When multiple virus amounts fulfill the above criteria, pick the amount of virus that gave the largest CTG Puro(+) value. This will usually match the peak of the Puro (+) CTG curve.
?. [Titration of Virus for Viability Assay]
Make a table (see below) and use this as a reference for the amount of virus to be used for cell lines with HIGH/MEDIUM/LOW infection sensitivity:  ABCDEFG1LacZCh2-2AAVS1...Target BTarget C2Low5 uL2.5uL10553Medium1052010104High2010402020
ABCDEFG1LacZCh2-2AAVS1...Target BTarget C2Low5 uL2.5uL10553Medium1052010104High2010402020
?. [7-Day Viability Assay]
Before starting the assy, you should know: Seeding Density and Infection Sensitivity (for each cell line tested) and Viral Titer (for each virus tested). (Day 0) Create a copy of the "CTG normalization template" and fill out the information (there are 6 lines per template; if testing > 6 cell lines, create multiple sheets).
?. [7-Day Viability Assay]
Cell seeding; seed # of cells in accordance with the result from the seeding density measurement; One 96-well plate (rows B-G; columns 1-11).
?. [7-Day Viability Assay]
(Day 1) Infection
?. [7-Day Viability Assay]
Dilute Virus with 10% FBS RPMI; use the "Virus Calculation for CTG Assay" template to calculate how much virus/medium to be mixed.
?. [7-Day Viability Assay]
Aliquot 325 uL of diluted virus to single row (columns 1-11)/cell line of the Master Plate(s).   ABCDEFGHIJKLM1Cell Line2ano infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-3bno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-4cno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-5dno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-6eno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-7fno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-8-------------
ABCDEFGHIJKLM1Cell Line2ano infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-3bno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-4cno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-5dno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-6eno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-7fno infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C-8-------------
?. [7-Day Viability Assay]
Replace the medium of cell-seeded plates with 50uL of 10% FBS RPMI containing 8ug/mL polybrene.
?. [7-Day Viability Assay]
Add 50 uL of virus from Master Plate to corresponding wells (in rows B-G) of cell-seeded plate.
?. [7-Day Viability Assay]
Spin plate at 900 x g for 1.5 hours at 37C.
?. [7-Day Viability Assay]
(for sensitive cells) replace medium with 100uL of fresh cell-specific medium right after spinning.
?. [7-Day Viability Assay]
(Day 2) Selection: Replace medium with 100 uL of fresh medium [for antibiotics (-)] or antibiotics-containing medium [for antibiotics (+)] .  ABCDEFGHIJKLM1-2(-)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C3(-)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C4(-)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C5(+)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C6(+)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C7(+)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C8-------------
ABCDEFGHIJKLM1-2(-)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C3(-)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C4(-)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C5(+)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C6(+)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C7(+)-no infectionsgLacZsgAAVS1sgCh2-2sgPolR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C8-------------
?. [7-Day Viability Assay]
(Day 5) Medium change: Replace medium with 100 uL of fresh medium [for antibiotics (-)] or antibiotics-containing medium [for antibiotics (+)]
?. [7-Day Viability Assay]
(Day 7) CTG Assay
?. [7-Day Viability Assay]
Final CTG values from a plate reader can be copied and pasted directly into "CTG Normalization Template. The results for the final assay will be normalized such that 0 is equal to an average of our negative (cutting) controls and -1 is equal to the average of our positive controls.
?. [Titration of Virus for Viability Assay]
(Day 0) Seeding: Prepare 15ml of cell suspension with:*[# of cells to seed per well] x 150 cells in R10, containing 10ul f 8 uG/uL polybrene
?. [Titration of Virus for Viability Assay]
Seed 100 uL/well into two 96-well plates per cell line:  ABCDEFGHIJKL1----no infectionsgLacZsgCh2-2sgAAVS1sgPOLR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C----2----100100100100100100100100100100ul3----100100100100100100100100100100----4----100100100100100100100100100100----5----100100100100100100100100100100----6----100100100100100100100100100100----7----100100100100100100100100100100----8------------------------------------------------  ABCDEFGHIJKL1----no infectionsgLacZsgCh2-2sgAAVS1sgPOLR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C----2----100100100100100100100100100100ul3----100100100100100100100100100100----4----100100100100100100100100100100----5----100100100100100100100100100100----6----100100100100100100100100100100----7----100100100100100100100100100100----8------------------------------------------------
ABCDEFGHIJKL1----no infectionsgLacZsgCh2-2sgAAVS1sgPOLR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C----2----100100100100100100100100100100ul3----100100100100100100100100100100----4----100100100100100100100100100100----5----100100100100100100100100100100----6----100100100100100100100100100100----7----100100100100100100100100100100----8------------------------------------------------
ABCDEFGHIJKL1----no infectionsgLacZsgCh2-2sgAAVS1sgPOLR2DsgSF3B1sgKIF11sgTarget-AsgTarget-BsgTarget-C----2----100100100100100100100100100100ul3----100100100100100100100100100100----4----100100100100100100100100100100----5----100100100100100100100100100100----6----100100100100100100100100100100----7----100100100100100100100100100100----8------------------------------------------------
?. [Titration of Virus for Viability Assay]
Make virus 'Master Plate':Create a master plate (enough virus for both seeded plates) by serial diluting pure virus in R10 with a multichannel pipette:  ABCDEFGHIJKL1------------------------------------------------2----Med150150150150150150150150150ul3----Med150150150150150150150150150----4----Med150150150150150150150150150----5----Med150150150150150150150150150----6----Med150150150150150150150150150----7----Med300300300300300300300300300----8------------------------------------------------Fill row G (columns 3-11) with indicated amount of virus. Fill rows B-F (columns 3-11) with indicated amount of medium (eg. R10). Fill rows B-F (columns 3-11) with indicated amount of medium (eg. R10). Transfer half the volume from row G to row F and mix well. transfer half the volume from row F to row E and mix well. Transfer half the volume from row E to row D and mix well. Transfer half the volume from row D to row C and mix well. Transfer half the volume from row C to row B and mix well.
ABCDEFGHIJKL1------------------------------------------------2----Med150150150150150150150150150ul3----Med150150150150150150150150150----4----Med150150150150150150150150150----5----Med150150150150150150150150150----6----Med150150150150150150150150150----7----Med300300300300300300300300300----8------------------------------------------------
?. [Titration of Virus for Viability Assay]
(Day 0) Infection: Transfer 40 uL of virus to cell-seeded plates form the corresponding well in Master Plate. Centrifuge at 900 x g, 37-degree for 1.5h.
?. [7-Day Viability Assay]
Replace medium with 100 uL/well of warm 10% FBS RPMI
?. [7-Day Viability Assay]
Add 25 uL/well of CellTiter-Glo Reagent (Promega, G7573)
?. [7-Day Viability Assay]
Shake on a plate shaker for 30 min RT
?. [7-Day Viability Assay]
Read luminescence using a plate reader (eg., EnVision, PerkinElmer, #2105-0010)