HSV Immunisation: Functional Analysis Post Castration

Contents:

 

 

 

 

Mice

Mouse groups:

  • Young - 2 month
  • Old - 2 year
  • Old Cx - Castrated 2 year-olds

These are all C57Bl6/J. Castrated mice will be surgically castrated following anaesthesia. Mice will be left until 6 weeks post-castration.

HSV Immunisation

  • Footpad immunisation of mice at 6 weeks post-castration. 5 x 10^6 PFU of HSV-1-KOS. 20uL injected into each footpad using a 30g needle.
  • D5 post-immunisation: remove both popliteal LNs from unimmunised, primary and secondary infected mice sterilly. Place into RPMI-10. Cell suspensions made by pushing through wire mesh with a syringe plunger.
  • Cell counts performed using Ethidium bromide/acridine orange.
  • 10^6 cells required for FACS analysis. 5-6 x 10^6 cells required for CTL assay.

 

FACS Analysis

  • Incubate 10^6 cells with aCD8-APC (CTLs), aCD25-PE (activated cells) and aVbeta10-Biotin (HSV specific).
  • Wash cells by addition of PBS/FCS/Az and centrifugation at 600g (max) for 5 mins.
  • Incubate with Strepavidin-CyChrome to reveal aVbeta10. Wash and resuspend in 200uL for FACS.

 

CTL Assay

5 x 10^6 cells cultured for 3 days (obtained at D5 post-immunisation).

Preparation of Standard tumour targets (EL4, E.G7, P815 etc.):

Targets should not be grown to densities greater than 10^6 cells/ml or spontaneous release will be unacceptable.

  • Pellet up to 1 x 10^6 EL4 cells in a 10mL centrifuge tube (x2).
  • Add: 150uL of complete media or RP10. Add 3 microlitres gB peptide to 1 tube. Then add 150uL of 51Cr [150 uCi] to both tubes.
  • Incubate for 45 minutes (can get away with as little as 30 minutes). Fill tube with media and invert to mix.
  • Pellet (wash 1) and aspirate the supernatant.
  • Resuspend in 10ml of media by flicking tube.
  • Add media to fill tube, make sure pellet is resuspended.
  • Pellet (wash 2) and discard supernatant.
  • Resuspend in 10ml media to give 1 x 10^5 cells/ml – targets are at 10^4 cells/well. Make sure pellet is well suspended by using 10ml ‘stripettes’.

 

Preparation of Effectors (on day of assay):

CTL effectors should be from day 3 bulk culture.

  • Count out cells as described in the formula below and pellet.
  • Starting at an E:T of 30:1 and with ‘n’ target wells, suspend:

(4.5n + 3) x 10 5 cells in (n x 150 + 100)uL

i.e. for 2 targets this is (9 + 3) = 12 x 10 5 cells/400uL

3 targets, (13.5 + 3) = 16.5 x 10 5 cells/550uL

4 targets, (18 = 3) = 21 x 10 5 cells/700uL

5 targets, (22.5 + 3) = 25.5 x 10 5 cells/850uL

 

  • Load 150uL in the top well (i.e: row 1) of a 96 well plate for each target/effector combination, then dilute 1/3 down, through rows 2 t0 6 using a twelve channel pipette (i.e: 50uL to 100uL of media and discard final 50uL). This leaves 100uL effectors/well.
  • Add 100uL of each target to the appropriate wells containing the effectors.
  • Add 100uL to four wells for each target: to two, add 100uL media (spontaneous), to the other two, add 100uL 1% triton (maximal).
  • Incubate for 4 to 6 hours at 37 0C/6.5% CO2.
  • Spin plates in IEC and collect 100uL of supernatant into the disposable small tubes. Place these into the larger reusable tubes and count.

 

Notes:

  • Assays are carried out in 96 well round-bottom plates.
  • Media used for CTL assays is RP5
  • Worthwhile counting cells after labelling.
  • Usually it is possible to get between 1-2 cpm/cell i.e: 5000-10000 cpm/100uL S/N.

 

Assay for Viral Clearance

A further group of mice will be analysed for their ability to clear viral particles.

 

  1. Sacrifice mice at D2 post-immunisation (as above); non-immunised, young immunised, old immunised and castrated old immunised mice.
  2. Remove feet above ankle. Place into RP10. Skin and tissue are removed from the bone and cut into small pieces.
  3. These are homogenised in a handheld homogeniser until an adequate cell suspension is achieved (make sure homogenisation is not too harsh as to cause cell death.
  4. Perform FACS analysis on a sample of cells to determine CD8 (CTL) T cell recruitment into the place of injection.
  5. Immediately freeze remaining cells at –70oC until required (in RP10). Note, freeze-thawing the cells will cause further viral release. The supernatant alone is required for the plaque assay.

PFU Assay

The assay is carried out in 6-23ll flat bottomed tissue culture plates containing a confluent monolayer of VERO cells.

 

  1. Prepare 1% agarose in MEM-2
  2. Make virus dilutions in a 24-well plate in serum-free MEM (SF-MEM) such that the virus is titrated from 10^-1 to 10^-8. Note: Each foot represents 1 sample. Use supernatant obtained after homogenisation.
  3. Remove supernatant from culture, confluent VERO cells CAREFULLY and wash x 1 with SF-MEM (to remove FCS) being careful not to disturb the monolayer. Carefully aspirate.
  4. For each virus sample, add 900uL virus dilution from wells 3-8 in the 24-well plate to the VERO cells in the 6-well plate.
  5. Incubate the 6-well plate for 1h at RT (in hood) with occasional shaking.
  6. Add 3 ml 1% agarose in MEM-2 (cooled to 40 0C) to each well. Swirl the plate immediately so the agarose and virus dilution mix.
  7. Allow the agarose mixture to set at room temperature. This will occur almost immediately.
  8. Incubate the plates in a 37 0C incubator for 3-4 days. Cheque for plaque formation under the microscope.

 

Fix and stain the VERO cells:

  1. Add 4ml/well of 10% formalin in phosphate buffer to the 6-23ll plates. This fixes the VERO cells and also kills the virus.
  2. Incubate plates at RT for one hour.
  3. Carefully remove formalin. Remove the agarose overlay using an 18G needle. Be careful not to scratch the cells with the needle.
  4. Add a few drops of 1% crystal violet to each well. Swirl the plates so that the crystal violet covers the well. Incubate for 1 min.
  5. Remove the crystal violet. Wash each well with water, swirl and remove excess.
  6. Repeat if necessary. Plaques should now be clearly visible.
  7. Let the plates dry upside-down on papertowels overnight.

 

Count the plaques:

  • Draw 6-8 lines across the back of wells that look like they contain 10-100 plaques.
  • Count the no. of plaques
  • Detemine the concentration of virus in your original sample:

Formula:

no. of plaques = pfu/ml

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Dilution x 0.9