sequence-specific primer (ssp) kit  (Thermo Fisher)

Journal: Virology Journal

Article Title: HLA-associated protection of lymphocytes during influenza virus infection

doi: 10.1186/s12985-020-01406-x

Figure Lengend Snippet: Protein Synthesis by Influenza Virus (IAV)-exposed Human PBMC. These representative images show results using cells from HLA-A2-homozygous donor number 1 in part b and HLA-A2-homozygous donor number 2 in part c. a Autoradiograms (monocytes/macrophages, lanes 1, 2; purified lymphocytes, lanes 3, 4) show representative protein synthesis results using PBMC from an HLA-A2-.heterozygous donor after sham-exposure or exposure of PBMC to IAV at an MOI = 10. Odd-numbered lanes show lysates of sham-exposed cells, and even-numbered lanes show lysates of IAV-exposed cells. HA = hemagglutinin, NA/NP = neuraminidase and nucleoprotein, which co-migrate, and M = the matrix protein. Numbers show positions of standard proteins having the indicated Mr. × 10 − 3 . b Autoradiograms show representative protein synthesis results using PBMC from an HLA-A2-homozygous donor. After sham-exposure (lanes 1, 2 and 5, 6) or exposure to IAV as PBMC at an MOI = 10 (lanes 3, 4 and 7, 8), purified monocytes/macrophages (lanes 1–4) and lymphocytes (lanes 5–8) were obtained and pulse-labeled and analyzed. Odd-numbered lanes show total cell lysates, and even-numbered lanes show lysates immunoprecipitated with mouse monoclonal anti-NP antibody. c Autoradiograms show cell lysates from an HLA-A2-homozygous donor (lanes 1–14) and an HLA-A1,2 donor (lanes 15–28) that were immunoprecipitated using mouse monoclonal anti-NP antibody and polyclonal anti-HA and anti-NA antibodies. After sham-exposure (odd-numbered lanes) or exposure to IAV (even-numbered lanes) as unseparated PBMC, purified lymphocytes and monocytes/macrophages were obtained, pulse-labeled and collected. Lymphocytes were pulse-labeled 0–2 h (lanes 1, 2 and 15, 16), 2–4 h (lanes 3, 4 and 17, 18), 4–6 h (lanes 5, 6 and 19, 20), 6–8 h (lanes 7, 8 and 21, 22), 8–10 h (lanes 9, 10 and 23, 24), and 22–24 h (lanes 11, 12 and 25, 26) after exposure. Monocytes/macrophages (lanes 13, 14 and 27, 28) were pulse-labeled 4–6 h after exposure. Lane 25 is blank (lysate not available). Lane 29 shows positions of standard Mr. proteins

Article Snippet: For a subset of donors, cellular DNA was extracted using the Easy DNA kit for genomic DNA isolation (Invitrogen, Carlsbad, CA) and HLA-A2 subtyping was performed using the Dynal HLA-A2 PCR sequence specific primers (SSP) subtyping kit (Lake Success, NY) [ ].

Techniques: Purification, Labeling, Immunoprecipitation

Journal: Virology Journal

Article Title: HLA-associated protection of lymphocytes during influenza virus infection

doi: 10.1186/s12985-020-01406-x

Figure Lengend Snippet: Influenza Virus (IAV) Neuraminidase (NA) RNA is Synthesized by HLA-A2- heterozygous (HTZ) but not HLA-A2-homozygous (HMZ) Lymphocytes. This representative image shows results using cells from HLA-A2-homozygous donor number 3. Autoradiograms of Northern blots show cell lysates from an HLA-A2-HMZ donor (lanes 1–10) and an HLA-A1,2 donor (lanes 11–20) obtained after sham-exposure (odd-numbered lanes) or exposure to IAV (even-numbered lanes). After exposure as unseparated PBMC, purified lymphocytes were obtained and lysates were collected after 2 h (lanes 1, 2 and 11, 12), 4 h (lanes 3, 4 and 13, 14), 8 h (lanes 5, 6 and 15, 16), and 24 h (lanes 7, 8 and 17, 18). Lysates of purified monocytes/macrophages (lanes 9, 10 and 19, 20) were collected 4 h after exposure. Lysates were probed for positive strand NA as described in the Methods. The original autoradiogram of the HLA-A1,2 donor’s lysates also showed a faint NA signal in lane 16 as well as 12 and 14

Article Snippet: For a subset of donors, cellular DNA was extracted using the Easy DNA kit for genomic DNA isolation (Invitrogen, Carlsbad, CA) and HLA-A2 subtyping was performed using the Dynal HLA-A2 PCR sequence specific primers (SSP) subtyping kit (Lake Success, NY) [ ].

Techniques: Synthesized, Northern Blot, Purification

Journal: Virology Journal

Article Title: HLA-associated protection of lymphocytes during influenza virus infection

doi: 10.1186/s12985-020-01406-x

Figure Lengend Snippet: Influenza Virus (IAV)-infected HLA-A2-homozygous Lymphocytes Serve as Infectious Foci for Uninfected Monocytes-macrophages. This representative image shows results using cells from HLA-A2-homozygous donor number 2. Autoradiograms show representative protein synthesis results using PBMC from an HLA-A2-homozygous donor. After sham-exposure (lanes 1 and 3) or exposure to IAV at an MOI = 10 (lanes 2 and 4), purified macrophages (lanes 1 and 2) and lymphocytes (lanes 3 and 4) were obtained and pulse-labeled. Additional aliquots of virus-exposed lymphocytes, after separation from macrophages by elutriation, were treated with neuraminidase, washed, and layered over additional aliquots of autologous control macrophages. After 1 h, the latter macrophages were extensively washed free of lymphocytes, pulse-labeled, and analyzed (lane 5)

Article Snippet: For a subset of donors, cellular DNA was extracted using the Easy DNA kit for genomic DNA isolation (Invitrogen, Carlsbad, CA) and HLA-A2 subtyping was performed using the Dynal HLA-A2 PCR sequence specific primers (SSP) subtyping kit (Lake Success, NY) [ ].

Techniques: Infection, Purification, Labeling

Journal: Virology Journal

Article Title: HLA-associated protection of lymphocytes during influenza virus infection

doi: 10.1186/s12985-020-01406-x

Figure Lengend Snippet: Influenza Virus (IAV)-infected Neuraminidase-treated HLA-A2-homozygous Lymphocytes Serve as Infectious Foci for MDCK Cells. This representative image shows results using cells from HLA-A2-homozygous donor number 2. Lymphocytes were layered over MDCK cell monolayers which were overlaid subsequently with 0.6% agarose, incubated at 37 °C for 48 h, then fixed and stained with methylene blue to facilitate plaque quantification. Based on previous studies, one plaque was assumed to be caused by one infected cell

Article Snippet: For a subset of donors, cellular DNA was extracted using the Easy DNA kit for genomic DNA isolation (Invitrogen, Carlsbad, CA) and HLA-A2 subtyping was performed using the Dynal HLA-A2 PCR sequence specific primers (SSP) subtyping kit (Lake Success, NY) [ ].

Techniques: Infection, Incubation, Staining

Journal: Virology Journal

Article Title: HLA-associated protection of lymphocytes during influenza virus infection

doi: 10.1186/s12985-020-01406-x

Figure Lengend Snippet: Influenza Virus (IAV)-specific Cytotoxicity is Related to HLA-A Determinants and Target Cell Populations. This representative image shows results using cells from HLA-A2-homozygous donor number 4. Cytotoxic T lymphocyte (CTL) activity was measured as specific lysis of autologous targets consisting of monocytes/macrophages (Δ), or lymphocytes/lymphoblasts (○), or unseparated PBMC (□). Results show virus-specific lysis of autologous target cell populations infected with the strain of virus used to induce CTL (A/Marton/43 H1N1). The graphs show CTL activity and autologous target cell susceptibilities for PBMC and subpopulations from ( a ) a heterozygous (A1,11) individual and ( b ) a homozygous (A2, −) individual. Results are representative of three experiments using PBMC from different heterozygous and homozygous donors tested concurrently

Article Snippet: For a subset of donors, cellular DNA was extracted using the Easy DNA kit for genomic DNA isolation (Invitrogen, Carlsbad, CA) and HLA-A2 subtyping was performed using the Dynal HLA-A2 PCR sequence specific primers (SSP) subtyping kit (Lake Success, NY) [ ].

Techniques: Activity Assay, Lysis, Infection