Solutions are usually stable for only 1C2?months at -20C. Ca3(PO4)2 transfection is based on forming calcium phosphate-DNA precipitates that facilitate DNA entry into target cells. (2020). Graphical Abstract Open in a separate window Before You Begin Biosafety The National Institutes of Health (NIH) recommendations classify HIV-1 like a Risk KPT-9274 Group 3 agent. Any study involving HIV-1-centered lentiviral vectors should be authorized by the institutional biosafety committee and carried out according to the recombinant DNA advisory committee (RAC) guidance. Additional details can be KPT-9274 found in the following document: We routinely use single round pseudoviruses (PVs) carrying a reporter gene to study HIV-1 envelope glycoprotein function. We prepare the PVs by co-transfecting cells with (1) a lentiviral vector that provides HIV-1 structural proteins and enzymes, (2) a firefly luciferase reporter vector, and (3) an envelope-expressing plasmid. Upon illness, the expression level of the Luciferase reporter protein in the infected cells gives an estimate of the degree of PVs illness. The flexibility of the system allows to combine the same core plasmids (1 and 2) with different viral envelope plasmids of interest. Thus, the system allows quick and exact measurements of specific viral envelope function. Cell Maintenance All cells should be passaged at least twice and should show healthy morphology before using them in the viral assays. We have not seen significant decrease in transfection efficiency (293T) or reporter activity (TZM-bl or Cf2Th-CD4/CCR5) even after 30 passages. We typically split the cells at 90% confluency (every 2C3?days) using the following protocol: (1) KPT-9274 Remove the media from your flask, followed by washing once with PBS. (2) Add 1C2?mL of dissociation reagent (TrypLE or StemPro Accutase) to the adhered cells and incubate until cells are detached (typically? 5?min). (3) Add 10?mL of DMEM KPT-9274 to the flask, collect the detached cells, and mix slowly by gentle pipetting up and down. (4) Dilute the cell suspension as necessary and transfer the required volume of cells to a new flask. All cells are typically managed in vented culture flasks at 37C, incubated with 5% CO2. gene (e.g., pHIVec2.luc). c. HIV-1-based packaging plasmid (e.g., psPAX2). 6. Plasmid DNA stocks are prepared by transformation of initial plasmids into chemically qualified DH5a or Stbl3 bacteria by standard warmth shock protocol. Mix & Go transformation kit (T3001; Zymo Research) can be used to avoid the need for heat shock, incubations, or outgrowth actions. Several commercial miniprep and midiprep packages are available from different vendors to purify plasmid DNA. Follow the manufacturers instructions. bacteria at a low-copy number and therefore the pHIVec2.luc plasmid should be prepared from a large volume culture. We usually grow the transformed bacteria in two to three 1-liter flasks made up of bacteria in 250?mL LB broth each. These solutions can be purchased commercially as a calcium phosphate transfection kit (e.g., K278001 from Invitrogen). pH is critical for transfection; all three buffers at different pH values should be tested by transfection of a plasmid made up of reporter gene (e.g., green fluorescent protein (gfp)) and measuring the expression level of the reporter protein or the efficiency of transfection. We routinely accomplish 90% transfection efficiency with plasmid made up of the gfp gene and optimal Ca3(PO4)2 reagents. Solutions are usually stable for only 1C2?months at -20C. Ca3(PO4)2 transfection is based KPT-9274 on forming calcium phosphate-DNA precipitates that facilitate DNA access into target cells. It is a quick, simple, and inexpensive method with high transfection efficiency of 293T cells. In our hands, Ca3(PO4)2-mediated transfection into 293T cells is as efficient as other commercially available reagents. TMB substrate can be purchased commercially (e.g., Pierce? TMB Substrate Kit Cat# 34021). Luciferase assay system that does not require the use of a luminometer with injectors can be purchased commercially (e.g., Bright-Glo? Luciferase Assay System from Promega). Ca3(PO4)2 transfection. a. Prepare 293T cells for transfection as explained in day 0 but incubate the plate/flask for only 4C5?h in tissue culture incubator at 37C and 5% CO2 concentration to allow the cells CLG4B to attach to the surface. b. Add chloroquine to a final concentration of 25M, incubate for 5?min, and prepare the Ca3(PO4)2 transfection combination. A typical template for preparing the transfection combination for any T-25 flask transfection is usually shown in Table 2. Add dropwise the transfection combination to the cells with gentle swirling. Table 2 A Template for Ca3(PO4)2-Mediated Transfection in a T-25 Flask We found that Ca3(PO4)2 transfection of 293T cells is usually more efficient 4C5?h after seeding the cells in comparison with 293T cells that were grown overnight (14C20 h) prior to transfection. According to our institutional biosafety approved protocol, at this point we transfer the plate/flask to our BSL2+ facility. Work at the BSL2+ facility follows BSL3 practices in a BSL-2 environment. The standard personal.