Methylcellulose for use in a gHV68 plaque assay (DL and EB)
- Autoclave 20g methylcellulose powder in a 1 liter bottle with a stir bar.
- Autoclave 500 ml millipore water.
(The above reagents can be made up ahead of time and stored at RT.)
- Methylcellulose, Sigma, M0387, 500g (1,500 cP)
- Using sterile technique, add 500 ml hot (near boiling) sterile water to 20g sterile (autoclaved) methylcellulose; this can be done as the water comes out of the autoclave, or if you've autoclaved your methylcellulose and water in advance, heat up the water for about 5 min in the microwave before adding it to the methylcellulose.
- Stir (with a stir bar or frequent rocking on bench) at RT until solution cools to approx 37C.
- At this stage, some people put the methylcellulose at 4C on a stirring plate overnight. This works fine if you don't need to use the methylcellulose until the following day. However, it will take longer in the next step to mix in the MEM if the methylcellulose is cool.
- After the methylcellulose has cooled to at least 37C, add 500 ml of 2x MEM. This should be done slowly and with mixing and it may require more time on a stir plate to get the medium to mix completely with the methylcellulose. As suggested above, the cooler the methylcellulose, the longer it will take to mix in the MEM.
- If necessary, warm the methylcellulose/media to 37C before layering over your plaque assay.
- Store the methylcellulose at 4C.
DCB secret method:
- Combine 500 ml boiling hot (5 min in microwave) sterile water and 20 g sterile MC in the hood.
- Mix well by occasionally shaking the bottle while allowing the colloidal solution cool to about 37-56C (cool enough not to denature the ingredients in the 2x MEM that you are about to add).
- Give the bottle one last hard shake to get the colloid as suspended as possible.
- In the hood, add 500 ml 2x MEM (premixed with Abx, FCS, glutamine and chilled to 4C) and mix well by shaking. The addition of the cold media will dissolve the MC immediately. Bubbles will rise and the solution will become clear.
- Store at 4C. Warm to 37C before use.
- The 2x MEM should include FCS, pen/strep and glutamine. The concentration of the FCS in 2x MEM should be 10% for plaque assays with gHV68 (final conc of the FCS in the methylcellulose solution will therefore be 5%). Other viruses may require different concentrations of FCS and other additives.
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