By Proudfoot, Amanda E.I.; Wells, Timothy N.C.; Power, Christine
Professional investigators describe intimately the main common innovations in chemokine biology. protecting either ligands and receptors, those with no trouble reproducible equipment conceal all facets of chemokine examine, starting from the cloning and characterization of chemokines and their receptors, by using animal types to review chemokine functionality in vivo. every one strategy additionally comprises proper history details, in addition to offering an invaluable bibliography that renders the research of chemokines available in any respect degrees of expertise. accomplished and hugely sensible, Chemokine Protocols deals experimental and medical chemokine researchers contemporary gold-standard number of confirmed tools for examining this biologically ubiquitous and significant type of proteins.
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Extra info for Chemokine Protocols (Methods in Molecular Biology Vol 138)
Remove the medium and unattached cells and replace with 5 mL of fresh TNM-FH/10% FCS. 8. Incubate at 27°C until the cells have formed 80–100% confluent monolayer. 2. Maintenance of Sf9 Cells 1. Remove medium and floating cells from a confluent monolayer. 2. Add 5 mL of TNM-FH,10% FCS to the 25-cm2 flask. 3. Gentry dislodge the cells by a cell scraper, and disperse by gently pipeting up and down several times with a 5-mL plastic disposable pipet. 4. Add 4 mL of TNM-FH/10% FCS to a fresh 25-cm2 flask for suspension cells and add 1 mL of the cell suspension (approx 106 cells/25-cm2 flask).
An optimal growth temperature is 27°C. In order to maintain consistency of cell growth and virus infection, cells should be used within 20–30 passages. After that, a culture should be replaced by retrieving a fresh batch of cells from liquid nitrogen. 2. To prevent accidental virus infection of cell cultures, all reagents should be separately prepared for noninfected cells and infected cells. 3. Although baculovirus gives a very restricted host range, wild-type and recombinant baculovirus should be treated as potential biohazards.
5. Heat-shock the mixture by placing the tube in 42°C water bath for 45 s. 6. Chill the mixture on ice for 2 min. 7. Add 900 µL of SOC medium to the mixture. 8. Place the tube in 37°C shacking incubator with gentle agitation and incubate for 4 h. 9. Spread 10 µL over a plate and 100 µL over another plate. 10. Incubate at 37°C for at least 24 h or until blue colonies are clearly identified. 3. Isolation of Recombinant Bacmid DNA 1. Select the largest and most isolated white colonies from the plates to avoid the selection of false positives and possible cross-contamination.