Functional Analysis Post Castration
- 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
- 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
- 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
- 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.
- 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.
5 x 10^6 cells cultured for 3 days (obtained at D5
Preparation of Standard tumour targets (EL4, E.G7,
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
- 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
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,
(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
- 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
- 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.
- Sacrifice mice at D2 post-immunisation (as above);
non-immunised, young immunised, old immunised and castrated old
- Remove feet above ankle. Place into RP10. Skin and tissue are
removed from the bone and cut into small pieces.
- 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.
- Perform FACS analysis on a sample of cells to determine CD8
(CTL) T cell recruitment into the place of injection.
- 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.
The assay is carried out in 6-23ll flat bottomed tissue
culture plates containing a confluent monolayer of VERO cells.
- Prepare 1% agarose in MEM-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.
- 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.
- 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.
- Incubate the 6-well plate for 1h at RT (in hood) with
- 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.
- Allow the agarose mixture to set at room temperature. This
will occur almost immediately.
- 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:
- Add 4ml/well of 10% formalin in phosphate buffer to the
6-23ll plates. This fixes the VERO cells and also kills the virus.
- Incubate plates at RT for one hour.
- Carefully remove formalin. Remove the agarose overlay using
an 18G needle. Be careful not to scratch the cells with the needle.
- 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.
- Remove the crystal violet. Wash each well with water, swirl
and remove excess.
- Repeat if necessary. Plaques should now be clearly visible.
- 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:
no. of plaques = pfu/ml
Dilution x 0.9