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Submission Info
Screening Agreement
Compound Submission Form

Screening Assays
-Biodefense Virus Panel
-BK Virus
-Hepatitis B Virus
-Hepatitis C Virus
-Herpesviruses
-Human Papilloma Virus
-Orthopoxviruses
-Respiratory Virus Panel
-SARS CoV

Data Interpretation
Medicinal Chemistry

Contact Info

C. Jonsson, Ph.D and M. Murray, Ph.D., Southern Research Institute
D. Smee, Ph.D., Utah State University

Hi/Lo Assay                   (Southern Research Institute)
Dose Response Assay    (Southern Research Institute)
CPE Assay                        (Utah State University)

SARS Hi-Lo Assay Protocol
(Southern Research Institute, C. Jonsson, M. Murray)

 NOTES

 ·         Cells are received in the morning, plated, and incubated overnight.

·         Batch size consists of 15 plates.  15 plates will be plated in the morning.

·         Drug additions are made to the cells the day after plating (4 plates at a time

·         All conditions must be kept sterile.

·         White clear-bottom plates are used for CellTiter Glo luminescence reads.

·         Black clear bottom plates are used for ATPlight luminescence reads.

REQUIRED MATERIALS/EQUIPMENT

A.  Equipment

• Titertek Multidrop 384 with stacker unit; under sterile hood
• 2 Multidrop cassette heads, sterile
• Biomek FX with carousel; under sterile hood
• Sorvall RT7 Centrifuge
• Drummond Pipettor
• Sterile tissue culture hood
• 3-M ORCA rail system (with Multidrop, WallacVictor, Lid Station, and Barcode Reader)
• Magnetic stir bars, small
• Stir plate
• 37^o C 5% CO₂ Incubator
• 37^o C Water Bath
• Timer

B.  Consumables

• 5, 10, 25, & 50 mL sterile disposable pipets
• 96-well Black and white tissue culture treated plates with barcodes
• 96-well V-bottom plates
• Corning 50 mL sterile centrifuge tubes
• Biomek P20 tips for FX (9 boxes/9 plates)
• Foil Plate Sealers
• 125- or 250-mL sterile disposable flasks, x2
• Disposable graduated cylinder
• Eppendorf tubes, sterile
• Gloves, Purple Nitrile
• Kimwipes, small
• Sterile disposable reservoirs
• Aluminum foil

C.  Materials

• 70% Ethanol
• Distilled water (dH₂O)
• Sterile water (filtered and autoclaved dH₂O)
• Project notebook

1.       Reagents

1.1.    P/S

·         Make 5.5 ml aliquots in sterilized eppie tubes, store at –20 C. 

·         Yield:  20x 5ml aliquots of P/S

1.2    L-Glutamine

·         Make 5.5 ml aliquots in sterilized eppie tubes, store at –20 C. 

·         Yield:  20x 5ml aliquots of L-Glut

1.3    FBS

·         FBS from Cell Biology should be heat inactivated for 30 min.

·         Make 50ml aliquots in sterilized eppie tubes, store at -20 C.

·         Yield: 10x 50ml aliquots of FBS

1.4    Media

Assay Medium: IMEM, 5%FBS, L-glut, antibiotics (w/o phenol red)

 25ml    Fetal Bovine Serum (Atlanta Biologicals Cat#S12450, Lot#B0053) Heat inactivated @ 56C for 30min.

   5ml    L-glutamine, 200mM [final conc. 2mM] (GibcoBRL Cat#25030-081, Lot31160728)

   5ml    Penicillin-streptomycin solution containing 1x10⁴ U/ml penicillin and 1x10⁴ ug/ml streptomycin [final conc. 100 U/ml penicillin and 100 ug/ml streptomycin] (GibcoBRL  Cat#15140-122, lot#1174381)

500 ml  DMEM; w/glutamine, w/o phenol red, gentamycin, proline (Cellgro; Cat#, Lot#)

1.5    CellTiter-Glo (See Promega for Complete Instructions)

·         Equilibrate Glo Buffer and Glo Substrate to room temperature prior to mixing. 

·         Glo substrate is found in –20 C floor freezer

·         Glo buffer is found in the 4 C walk-in cooler

·         For the smaller Glo substrate bottles (size A), transfer 10 ml of Glo Buffer into the amber bottle containing the substrate.

·         For the larger Glo substrate bottles (size B), transfer 100 ml of Glo Buffer into the amber bottle containing the substrate.

·         Recap with rubber cap and mix gently, inverting ~10-20 times to dissolve all the substrate.

·         Transfer the solution into a sterile graduated cylinder.

1.6    1% DMSO in media (for validation; 1 drug plate is enough for 9 cell plates)

·         Add 19.8 ml of sterile dH₂O into a 125 ml flask.

·         Add 0.2 ml of DMSO.

·         Pour into a sterile reservoir.

·         Aliquot 200 ul/well into a sterile 96 V bottom plate, columns 1-11.

·         In a separate sterile 96 well V bottom plate aliquot 200 ul/well into column 12 only.

Table 1:  96-well Z-plate format

Table 2:   96-well plate format