Papillomavirus Inhibition Assay

Laboratory of Cellular Oncology, NCI

Purpose: screen compounds of interest for potential to inhibit HPV infectivity.

1)  Preplate HeLa cellsin 96-well plates.  Deliver 50 µl of cells suspended at 120,000 cells/ml to each well using a multichannel pipettor.  Incubate overnight.

2)  Dissolve candidate microbicidesat 1 mg/ml in water (or appropriate solvent).  For inexpensive/abundant substances, tare the lid of a 50 ml conical tube on the Mettler balance.  Tap 10 – 50 mg of substance into the center of the lid.  Screw the tube onto the lid, tap the tube, then add an appropriate number of mls of sterile water.

3)  Dilute microbicides.  Perform 3-fold serial dilutions in a U-bottom untreated 96-well plate using a multichannel pipettor moving from left to right.  The first well should have 100 µl of DMEM-10 + 45 µl of microbicide stock + 5 µl of 10x PBS (=300 µg/ml, will be 100 µg/ml final). Subsequent wells should have 100 µl of DMEM-10.  Use the multichannel pipettor to mix the sample then transfer 50 µl to the next well. Change tips in between dilutions. Make a total of 10 dilutions for each microbicide, covering final concentration ranging from 100 µg/ml to 5 ng/ml.  11^thand 12^thcolumns of wells should have medium with no microbicide.  If WST-1 assay will be performed (step 8), leave several outer wells with no cells.

• Note:  Ideally, it is best to avoid using the outermost wells of the 96-well plate, since they are prone to evaporation (so-called “edge effects”).  It’s OK to use HeLas in the outer wells as a “no virus” control, provided the wells have been fed on the same schedule as other wells.

4)  Add microbicides to cells.  Use a multichannel pipet to transfer 50µl of each microbicide dilution to the HeLa plate.  It’s OK to use the same set of tips going from most dilute to least dilute condition (i.e., moving from right to left)

5)  Add pseudovirus.  Dilute HPV16 pseudovirus stock into DMEM-10 supplemented with 3x pen-strep-fungizone(CB has frozen 100x PSF aliquots).  For an HPV16 stock with a standard 293TT titer of 5 x 10⁹, dilute stock roughly 1:15,000 (i.e., deliver ~0.003 µl of stock per well (~1 ng/ml L1 final)).  Place diluted pseudovirus in a sterile basin.  Distribute 50 µl of diluted pseudovirus stock to appropriate wells. Add 100 µl of plain DMEM-10 to any outer wells to be used as “no virus” control.

6) Add 100 µl of DMEM-10 ~24 hours after virus inoculation.

7) Incubate cells a total 48-56 hours after virus inoculation.

8)  (Optional):  Assess cytotoxicity. Inspect cells under microscope.  Aspirate medium using an 8-channel manifold (CLP #9621) attached to a 10ml pipet.  Be sure to remove the cotton ball from the pipet before aspirating (aspirated cotton balls will clog the vacuum trap).  Wash cells once with FACS wash medium (DPBS with calcium and glucose (Invitrogen# 14287-080) + 1% FCS + 1% Hepes + 1% PSF).  Remove wash and add 50µl of neutralization assay mediumsupplemented with 5% WST-1 metabolic substrate (Roche).  Return plate to incubator for ~20 minutes.  Read absorbance at 450 nm (reference 650 nm) on an ELISA plate reader.

9)  Trypsinize cells. Aspirate medium (see step 8). Add 50 µl of 0.05% trypsin / EDTA to all wells.  Aspirate trypsin.  Add another 50µl of trypsin, then incubate at 37ºC for 10 minutes, tapping plate occasionally.  

10)  Resuspend cells for FACS.  Using a multichannel pipettor, add 200µl of FACS wash medium (see step 8) to each well.  Triturate (pipet up and down 5-8x) to achieve a single-cell suspension.  Transfer 200 µl of cell suspension into pre-racked 1.2 ml vials (USA Scientific# 1265-0400, above ELISA bench).  Although it’s OK to use the same set of pipette tips if going from the most concentrated to least concentrated wells (i.e., from left to right - minimizes the possibility of carryover of GFP⁺cells into GFP^-wells), I usually use a 12-channel pipettor and just change tips between rows.

12)  FACS cells by nesting each 1.2 ml vial into a 5 ml Falcon FACS tube.  Collect 10,000 events.

Alternative procedure for handling extremely limited amounts of candidate compound:  test five doses of microbicide by adding stock directly to individual wells.  Add 10, 3.3, 1.1, 0.37 and 0.15 µl to each of five wells.  Note that there will be variability in the last dose, even when using a 2 µl pipettor with short micro tips.  Compounds determined to have effective doses in this range should be re-tested.  Pseudovirus can be added as 1µl of a 1:20 dilution.