Journal: Frontiers in Immunology

Article Title: Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues

doi: 10.3389/fimmu.2023.1213180

Figure Lengend Snippet: Correlating changes in NADH and eGFP expressing HIV-1 infection. 2p-FLIM imaging of eGFP and NADH with MOLT-4/CCR5 cells upon infection with HIV-1 eGFP virions. Differences in eGFP fluorescence intensity (A, B) , NADH fluorescence intensity (C, D) , average FLT of NADH (E, F) , and bound NADH FLT (G, H) for MOLT-4/CCR5 cells infected with virions compared to MOLT-4/CCR5 control. Close-up views of infected cells are shown in the panels adjacent to (B, D, F, H). Intensity histograms from corresponding images from cells with eGFP (I) and NADH (J) are shown. (K) shows the correlation plot between eGFP and NADH from individual cells. (L) shows the intensity decay of NADH from HIV-1 infected cells. The dots represent the experimental data and solid line is the bi-exponential fit to the intensity decay (see methods). Average FLT and bound form of NADH FLTs are quantified in bar graphs (M, N) . All experiments were repeated, with the average values shown. Error bars indicate standard deviations. Two-tailed test was performed for statistical analysis in panels (M) and (N) , P values; * <0.05, ** <0.01.

Article Snippet: Commercial cell lines THP-1 and MOLT-4/CCR5 obtained from ATCC are provided with certification that they are Mycoplasma -free.

Techniques: Expressing, Infection, Imaging, Fluorescence, Two Tailed Test

Journal: Nature Microbiology

Article Title: Monocyte-derived macrophages contain persistent latent HIV reservoirs

doi: 10.1038/s41564-023-01349-3

Figure Lengend Snippet: a , To determine the appropriate expander cell line, one viremic vPWH was activated with PMA in the context of MT-4, Molt-4-CCR5, CEMx174 and media only, dotted line indicates limit of detection (LOD). b , To determine the best activation condition, 7 participants ( n = 7, 4 vPWH and 3 virally suppressed vsPWH) were activated with PMA, IL-4, TNF𝛼 and media with and without MT-4 expander cells; mean ± s.d. c , To determine whether macrophages derived from negatively selected monocytes could be reactivated similarly to macrophages derived from whole PBMCs, we compared 3 participants ( n = 3, 1 vPWH and 2 vsPWH) activated with PMA and co-cultured with MT-4. d – f , To determine the appropriate assay to detect T cell contamination, in the well or via phagocytosis, we assessed CD3ε and TCRβ RNA expression in CD4 T cells isolated from healthy donors (HD). d , The CD4 T cells from 3 HD were serially diluted, lysed and RNA extracted to measure TCRβ and CDε expression; mean ± s.d. TCRβ ( e ) and CDε ( f ) showed similar variability across replicates, except at the low end of the assay. g , CD4 T cells were isolated from 8 HD; 1 × 10 6 CD4s per donor were lysed and assessed for CD3ε and TCRβ expression to determine the copies of each per cell; bar indicates median value. h , Healthy MDMs were co-cultured with HIV+ CD4 T cells from two donors (CP11 and 21) with and without PMA activation to determine whether HIV+ CD4 T cells were able to transfer viral nucleic acids to MDM; n = 2. i , A schematic of the final MDM-QVOA experimental design.

Article Snippet: Three lymphocyte cell lines were tested during the development of the MDM-QVOA: MT-4 cell line obtained through the NIH HIV Reagent Program, Division of AIDS (NIAID, NIH: MT-4 cells, ARP-120, contributed by Dr Douglas Richman (cat. no. 120) – ); MOLT-4-CCR5 kindly donated by Dr Robert F. Siliciano from Johns Hopkins Medical School; and CEMX174 purchased from ATCC.

Techniques: Activation Assay, Derivative Assay, Cell Culture, RNA Expression, Isolation, Expressing

Journal: Nature Microbiology

Article Title: Monocyte-derived macrophages contain persistent latent HIV reservoirs

doi: 10.1038/s41564-023-01349-3

Figure Lengend Snippet: Cell lines, MT-4, CEMx174 and MOLT4-CCR5 were assessed for HIV entry receptor expression CCR5, CXCR4 and CD4. Cell were gated on singlets and then live cells, shaded histogram is unstained cells, black line marker of interest.

Article Snippet: Three lymphocyte cell lines were tested during the development of the MDM-QVOA: MT-4 cell line obtained through the NIH HIV Reagent Program, Division of AIDS (NIAID, NIH: MT-4 cells, ARP-120, contributed by Dr Douglas Richman (cat. no. 120) – ); MOLT-4-CCR5 kindly donated by Dr Robert F. Siliciano from Johns Hopkins Medical School; and CEMX174 purchased from ATCC.

Techniques: Expressing, Marker

Journal: Nature Microbiology

Article Title: Monocyte-derived macrophages contain persistent latent HIV reservoirs

doi: 10.1038/s41564-023-01349-3

Figure Lengend Snippet: Culture supernatants from CP11 ( A ), CP21 ( B ), CP25 ( C ), and CP36 ( D ) positive MDM and CD4 QVOA wells were used to infect MT-4 cells and viral kinetics were assessed by measuring HIV RNA in supernatant overtime.

Article Snippet: Three lymphocyte cell lines were tested during the development of the MDM-QVOA: MT-4 cell line obtained through the NIH HIV Reagent Program, Division of AIDS (NIAID, NIH: MT-4 cells, ARP-120, contributed by Dr Douglas Richman (cat. no. 120) – ); MOLT-4-CCR5 kindly donated by Dr Robert F. Siliciano from Johns Hopkins Medical School; and CEMX174 purchased from ATCC.

Techniques: