Human Retrovirus Protocols: Virology and Molecular Biology by Tuofu Zhu

By Tuofu Zhu

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Extra resources for Human Retrovirus Protocols: Virology and Molecular Biology (Methods in Molecular Biology 304)

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18. Centrifuge tubes with PBL or monocytes at 400g for 10 min at room temperature. Decant the supernatant and resuspend the cells in 25 mL of PBS. 19. Use a small aliquot (10 mL) to count the cells and calculate the total number of cells. 20. To check the purity of PBL and monocyte populations, make a cell smear on a glass slide using a small aliquot from each cell suspension and stain with hematoxylin and eosin (H&E). Observe under the light microscope. Figure 2 shows H&E-stained monocytes and PBL separated by this procedure.

4. 5. 6. 7. 8. Prepare the elutriation setup before starting to process the blood sample. Assemble the chambers and the rotor per the instrument instruction manual. Connect the input tubing and the output tubing to the rotor chamber. The input tubing is connected through a peristalitic pump that controls the flow rate. To the other end of the input tube connect a three-way stopcock, which is used to control the input of either PBS or cells pumped from two separate reservoirs (the manual provides detailed instructions for the setup).

3. Methods The methods described below outline (1) the isolation of peripheral blood mononuclear cells from patient blood and buffy coat, (2) the virus isolation from patient PBMC in bulk, (3) HIV isolation under limiting dilution conditions to obtain biological virus clones, (4) the propagation of primary HIV isolates and variants, and (5) the titration of virus stocks (see Note 1). 1. Isolation of PBMC From Patient Blood and Buffy Coats Transfer heparinized venous patient blood to plastic 50-mL tubes (maximum of 25 mL patient blood per tube) and dilute with an equal volume of PFH medium.

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