Protocol for activator screen

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Remove B1-B62 from freezer and place inside hood

Prewarm RPMI 1640 medium (1-2 tubes)
Put PMA on ice
Pipet 100 microL of medium into 64 wells on a 96 well clear bottom black
face plate
5. Pipet 0.8 microL of compounds B1-61 into 62 wells
6. Pipet 0.8 microL of PMA into 1 well
7. Sit for 20 mins (dark)
8. Measure cell viability (J-Lat Full) prior to changing medium
a. T: []cells/mL
b. L: []cells/mL
c. V: []%
9. Determine volume from cell stock: mL
a. V = (10^5 * 80 wells) / # of live cells/mL
i. E.g. 1.57*10^6 live cells/mL
ii. (10^5*80)/( 1.57*10^6 live cells/mL) = 5.10 mL
10.Remove volume (determined in step 4) from cell stock and pipet into test
tube.
11.Spin down test tube 300G * 6 minutes
12.Discard supernatant
13.Pipet fresh medium and mix to break cell pellet (use same medium used for
compounds)
a. Volume of medium = # wells * 100 microLiter
b. E.g. 80 wells * 100 microLiter = 8 mL
14.Put cells + fresh medium into reservoir
15.Pipet 100 microL of cells + medium into 64 wells
16.Place wells into incubator
17.Note Time: []
Stimulation of cells with Ad5 and MVA
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Prewarm RPMI 1640 medium (1 tubes)

Remove Ad5 and MVA antigens from -80 freezer
Label 3 wells of a 24 well flat bottom plate as NC, Ad5, MVA
Pipet 300 uL of RPMI medium into 3 wells (NC, Ad5, MVA)
Pipet Ad5 and MVA into 2 wells (Ad5, MVA) at 3 MOI
a. .4 uL of Ad5 (Pipet 1uL of Ad5 with 9uL of medium; pipet 4uL of
solution into well)
b. 3.4 uL of MVA
6. Measure cell viability of THP-1 Cells
a. T: []cells/mL
b. L: []cells/mL
c. V: []%
7. Calculate [] mL of THP-1 cells required from stock
a. You want: 4x10^5 cells / well
b. You have: 4 wells (3 wells + 1 extra well for good measure)
c. Total Cells: 4 * 4*10^5 cells = 1.6*10^6 cells
d. 1.6*10^6 cells/L:[]cells/mL = [] mL from stock
8. Remove [] mL of THP-1 cells from stock and place into new tube

9. Centrifuge 300g x 6 mins

10.Discard supernatant
11.Resuspend cell pellet in [4 wells] * [200]uL = 800uL of fresh medium
12.Pipet 200 uL from cells + new medium in each well; discard extra
13.Incubate [] time
RNA Extraction
1. Label 1.5 mL tubes NC, Ad5, MVA (create 3 sets = 9 tubes total)
2. Label RNA IC tube + Collection Tube: NC, Ad5, MVA (3 tubes)
3. Resuspend cells in plate
4. Transfer cells from plate to 1.5mL tubes
5. Centrifuge 300g x 6 mins
6. Discard supernatant
7. Resuspend cell pellet in 300 mL RNA lysis buffer
8. Sit for 10 mins
9. Centrifuge at 12,000 rpm x []mins
10.Transfer supernant to New tubes
11.Add equal volume (300mL) of ethanol to tubes; mix well
12.Follow RNA prep kit protocol (make sure to skip that one step)
RNA Detection
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Turn on microplate reader

Prepare plate
A row blank
B1 B2 B3: NC, Ad5, MVA
Pipet 2 uL onto spot; close plate
Place plate onto reader
Run template protocol
Results: RNA Concentration

Reverse Transcription
1. Prepare 3 RT tubes (super small) in air flow hood. Label NC Ad5 MVA
2. Calculate 900 / RNA concentration = [] uL of RNA for each (NC, Ad5, MVA)
3. For each tube:
a. 15 - [] uL of RNA for each = uL of dH20
b. 4uL of mix
c. 1uL of RTase (add last)
d. [] uL of RNA for each
e. Total volume = 20uL
4. Place the 3 tubes into the RT machine
5. Select RT protocol (95 C in it)
6. Select run (approx. 1 Hr)
7. Freeze 1.5 mL tubes with extra RNA that was extracted (remaining volume
15-2uL to pipet in reader-[] uL of RNA = remaining amount in tube)
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