Journal: Environmental Science & Technology

Article Title: Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge

doi: 10.1021/acs.est.3c02381

Figure Lengend Snippet: Visualization of PHA accumulation in activated sludge by staining PHA with BODIPY (green) and protein SYPRO Red (red). Samples at the start of accumulation (a–c) and after 24 h of accumulation (d–f). Scale bars represent 10 μm.

Article Snippet: BODIPY 493/503 (BODIPY) (Thermo Fisher Scientific, MA) for PHA granule staining was combined with protein staining via SYPRO Red originally 5000× concentrated (Thermo Fisher Scientific, MA).

Techniques: Staining

Journal: Environmental Science & Technology

Article Title: Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge

doi: 10.1021/acs.est.3c02381

Figure Lengend Snippet: Direct accumulation replicate experiments with developments over 48 h revealed by combined PHA (green) and protein (red) staining. Yellow represents the overlapping signal between PHA and protein staining. Samples from replicate accumulations ACC 1 (a–d), ACC 2 (e–h), and ACC 3 (i–l). Scale bars represent 10 μm.

Article Snippet: BODIPY 493/503 (BODIPY) (Thermo Fisher Scientific, MA) for PHA granule staining was combined with protein staining via SYPRO Red originally 5000× concentrated (Thermo Fisher Scientific, MA).

Techniques: Staining

Journal: Environmental Science & Technology

Article Title: Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge

doi: 10.1021/acs.est.3c02381

Figure Lengend Snippet: Direct accumulation replicate experiments with developments over 48 h revealed by combined PHA (green), DNA (blue), and FISH (red). Magenta represents the overlapping signal between RNA and DNA staining. Yellow represents the overlapping signal between RNA and PHA staining. Samples from replicate accumulation ACC 1 (a–d), ACC 2 (e–h), and ACC 3 (i–l). Scale bars represent 10 μm.

Article Snippet: BODIPY 493/503 (BODIPY) (Thermo Fisher Scientific, MA) for PHA granule staining was combined with protein staining via SYPRO Red originally 5000× concentrated (Thermo Fisher Scientific, MA).

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates

doi: 10.3389/fimmu.2023.1188018

Figure Lengend Snippet: Production of conserved elements (CE) antigen-specific nonhuman primate T-cells (NHP CE-XTCs) from vaccinated macaques. (A) Macaques received a CE DNA vaccine on vaccination weeks 0 and 4 (open triangles) followed by two additional doses of the CE DNA vaccine co-delivered with a second plasmid expressing full length (FL) SIV Gag and Env on weeks 8 and 20 (closed purple triangles). NHP CE-XTC expansion began 2 weeks after the final CE vaccine dose with 30-50 mL of peripheral blood. Schedule is also shown relative to weeks post-SHIV infection (“SHIV Week”) and post-antiretroviral therapy (ART) initiation (“ART Week”). (B) Following Ficoll separation of week 22 blood draws and a plastic adherence step, the non-adherent fraction was cryopreserved, and an 8-day dendritic cell (DC) culture was initiated (blue bar). NHP CE-XTCs (red bar) were induced by mixing with autologous, CE peptide-pulsed, irradiated DC. DC manufacturing spanned Days -8 through 0, and were added to CE-XTCs on Day 0. Non-adherent PBMC stimulated with phytohemagglutinin (PHA) and IL2 (PHA blasts, green bar) were similarly pulsed with CE peptides and irradiated on Day 11. Finally, GM-K56 cells, typically expanded over a 2-week time course (orange bar), were irradiated and added to the culture along with PHA blasts on Day 11. Day 11 CE-XTC cultures were mixed with irradiated, peptide-pulsed PHA blasts and irradiated GM-K562 feeder cells at a 1:1:4 ratio. This mixed culture was rapidly expanded in G-Rex 100 flasks for at least one week prior to infusion.

Article Snippet: Base media for CE-XTC and PHA Blasts was 45% Advanced RPMI (Thermo Fisher), 45% Eagle Hank’s Amino Acids Click’s (Irvine Scientific, Santa Ana, CA), and 10% heat-inactivated Human AB serum (Gemini Bio, West Sacramento, CA) plus 2mM GlutaMax.

Techniques: Plasmid Preparation, Expressing, Infection, Irradiation

Journal: Frontiers in Immunology

Article Title: Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates

doi: 10.3389/fimmu.2023.1188018

Figure Lengend Snippet: Ex vivo expansion of NHP CE-XTCs and supporting cells. NHP CE-XTCs were prepared as described in Figure 1 and cell counts at each manufacturing phase were determined from SHIV-infected, ART suppressed donors (closed circles, n = 5) and uninfected donors (open circles, n = 2). (A) Cell counts from representative manufacturing time points detailed in Figure 1 , including initiation of DC culture (Day -8 DC), CE-XTCs on Day 0 and Day 11 following addition of CE-peptide pulsed, irradiated DC, PHA blasts on Day 4 (thaw day) and Day 11 (after 7 days of expansion), and co-cultured CE-XTC on Day 11 (addition of expanded PHA blasts and GM-K562) and Day 17 (after 6 days of expansion). “CE-XTC + PHA Blasts + GM-K562” includes comparisons of cell numbers from SHIV-infected, ART-suppressed donors at day 11 and 17 when expanded in the absence (-ART) or presence (+ART) of antiretroviral drugs in the culture media. (B) Fold expansion for the CE-XTC, PHA blasts, and CE-XTC/PHA blast/GM-K562 mixed cultures in the absence (-ART) or presence (+ART) during the indicated time interval. C:P:G refers to the final co-culture of CE-XTC, PHA blasts, and GM-K562 feeders. Cells from uninfected donors (animal IDs A17044 and A17045) were only assessed in the absence of ART. In subsequent figures, CE-XTC phenotypic and functional data includes 3 of 5 SHIV-infected, ART-suppressed donors (IDs A17026, A17032, and A17035) but was not available from 2 others (IDs A17023 and A17025).

Article Snippet: Base media for CE-XTC and PHA Blasts was 45% Advanced RPMI (Thermo Fisher), 45% Eagle Hank’s Amino Acids Click’s (Irvine Scientific, Santa Ana, CA), and 10% heat-inactivated Human AB serum (Gemini Bio, West Sacramento, CA) plus 2mM GlutaMax.

Techniques: Ex Vivo, Infection, Irradiation, Cell Culture, Co-Culture Assay, Functional Assay