By Tuofu Zhu
A state-of-the-art selection of easy and state of the art tools optimized for investigating the molecular biology of this type of retrovirus. those effortlessly reproducible concepts diversity from tools for the isolation and detection of human retroviruses to state of the art tools for exploring the interaction among the viruses and the host. right here, the researcher will locate up to date innovations for the isolation and propagation of HIV, HTLV, and foamy virus from various assets. There also are assays for choosing the phone tropism of HIV-1, the coreceptor utilization of HIV-1, and human gene expression with HIV-1 an infection via microarrays, in addition to for phenotyping HIV-1 contaminated monocytes and reading their health. Highlights contain the detection and quantification of HIV-1 in resting CD4+, a brand new cloning process for making recombinent virus, cDNA microarrays, and the decision of genetic polymorphisms in lately pointed out HIV-1 co-factors which are serious for HIV-1 an infection.
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Extra info for Human Retrovirus Protocols: Virology and Molecular Biology (Methods in Molecular Biology)
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.
304: Human Retrovirus Protocols: Virology and Molecular Biology Edited by: T. , Totowa, NJ 17 18 Schuitemaker and Kootstra 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Trisodium citrate dihydrate (TNC, Merck). Phosphate-buffered saline (PBS). Fetal calf serum (FCS; Hyclone). Recombinant interleukin-2 (rIL-2; Chiron Benelux). Penicillin (Pen), 100 U/mL (GibcoBRL). Streptomycin (Strep), 100 U/mL (GibcoBRL). Phytohaemagglutinin (PHA; Wellcome). Heparinized peripheral blood or buffy coat.
4. In some isolation attempts, very low concentrations of HIV-1 may be recovered; in these cases, the concentration of p24 antigen in culture supernatants may be below the limit of detection of commercial kits. The occurrence of undetectable p24 antigen in culture supernatants is more frequent in isolations from monocytes of patients undergoing HAART with plasma HIV-1 RNA levels below detection, compared to untreated viremic patients or patients failing HAART. 5 kit because of the ultrasensitivity of this assay.