February 22, 2017

A Note on Two Dogmas in Pragmatics by Andrzej Boguslawski

By Andrzej Boguslawski

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Remove caps and lower the level of fluid to just above the specimen. Allow the propylene oxide to evaporate from the vials for at least 2 hr. Infiltrate with 1 ml 100% epoxy resin (EMBED 812) for 2 hr or longer. Prepare a conical BEEM capsule for each specimen, and insert a paper label. Place a small drop of fresh epoxy resin in each capsule; close the cover. Lightly lubricate each capsule with silicon grease and press into the top of a microfuge tube (remove the cover of the microfuge tube). Spin the capsules for 5 sec to pull the epoxy resin to the tip of the capsule.

Cells are split 1 : 4 and maintained in a humidified 37°C incubator containing 5% CO,. Routinely, we plate MDBK cells on 100-mm tissue culture dishes (No. 3 100; Costar, Cambridge, MA) for large-scale preparation of virus stocks. 3. 5 ml 1 : 1000 dilution of virus stock. Virus stocks usually have a titer of at least 1-5 x lo8 pfu/ml which, when diluted 1 : 1000, gives a titer 1. Viruses as Model Systems in Cell Biology 17 of 1-5 x lo5pfulml. 5 x lo7cells. 05 pfulcell) to avoid problems caused by DI particles (see preceding discussion).

6. Purification of Virus 1 . To prepare purified influenza virions, MDBK cells (1 00-mm dishes) are inoculated at a multiplicity of 1-10 pfu/cell. The cells are incubated in a humidi- 24 Richard W. Compans and Paul C. Roberts fied 37°C chamber under an atmosphere of 5% CO, for 2 hr with periodic tilting. 2. The virus inoculum is removed and 6 ml Dulbecco’s reinforced Eagle’s medium with 2% CS is added per plate. The medium from injected plates is collected 24-36 hr postinfection and is precleared to remove cell debris by centrifugation at 5,000 g for 20 min.

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