Vectors were generated by triple transfection of HEK293 cells as described previously [116 (link)]. Cells were cultured in roller bottles (Corning) in DMEM 10% FBS to 80% confluence and co-transfected with a plasmid carrying the expression cassette flanked by the AAV2 viral ITRs, a helper plasmid carrying the AAV rep2 and cap9 genes, and a plasmid carrying the adenovirus helper functions (kindly provided by K.A. High, Children’s Hospital of Philadelphia). The expression cassettes used were one of the following: eGFP under the control of the CMV promoter, eGFP under the control of the CAG promoter, or murine Tert under the control of the CMV promoter. AAV9 vectors were purified with an optimized method based on two consecutive cesium chloride gradients, dialyzed against PBS, filtered, and stored at -80 °C until use [117 (link)]. The titer of the viral genome particles was determined by quantitative real time PCR.
Roller bottle
Manufactured by Corning
Sourced in United States
Roller bottles are a type of cell culture vessel used for the growth and propagation of cells. They provide a larger surface area compared to traditional cell culture flasks, allowing for increased cell yields. Roller bottles are designed to be rotated along their longitudinal axis, enabling uniform distribution of the cell culture media and ensuring efficient gas exchange. This equipment is commonly used in various applications, such as vaccine production, protein expression, and tissue engineering.
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Lab products found in correlation
Accutase, by Thermo Fisher Scientific (4 mentions)
Middlebrook 7h11 plates, by BD (2 mentions)
Tween 80, by Merck Group (2 mentions)
Middlebrook 7h9 broth, by BD (2 mentions)
Glycerol, by Carl Roth (1 mentions)
Thy broth, by Thermo Fisher Scientific (1 mentions)
Yeast extract, by BD (1 mentions)
Glycerol, by BD (1 mentions)
Dubos medium, by BD (1 mentions)
Glycerol, by Merck Group (1 mentions)
Mouse monoclonal antibody isotyping kit, by Merck Group (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Innova 4000 benchtop incubator shaker, by Eppendorf (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Vr 105, by American Type Culture Collection (1 mentions)
Soybean peptone, by Merck Group (1 mentions)
Maxq 6000, by Thermo Fisher Scientific (1 mentions)
Atcc mya 3404, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Biowest (1 mentions)
19 protocols using roller bottle
1
AAV9 Vector Production in HEK293 Cells
2
AAV9-Mediated Telomerase Gene Delivery
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Viral vectors were generated by triple transfection of HEK293 cells and purified as previously described (Matsushita et al., 1998 (link); Ayuso et al., 2010 (link)). Cells were cultured in roller bottles (Corning) in DMEM supplemented with 10% FBS to 80% confluence and cotransfected with a plasmid carrying the expression cassette flanked by the AAV2 viral inverted terminal repeats, a helper plasmid carrying the AAV rep2 and cap9 genes, and a plasmid carrying the adenovirus helper functions (plasmids kindly provided by K.A. High, Children’s Hospital of Philadelphia, Philadelphia, PA). The expression cassettes were under the control of the cytomegalovirus promoter and contained a SV40 polyA signal for eGFP, and the cytomegalovirus promoter and the 3′-untranslated region of the Tert gene as polyA signal for Tert. AAV9 particles were purified using two cesium chloride gradients, dialyzed against PBS, filtered, and stored at −80°C until use. Then, 27–30-wk old Tert+/+ and G3 Tert−/− mice were administered with 2E12vg of AAV9-Tert and AAV9-empty virus particles in a volume of 100 µl 0.001% Pluronic F-68 in PBS 1X by a single intravenous tail injection (Fig. 6 A ). Pluronic F-68 was used to prevent aggregations of the virus particles (Foust et al., 2009 (link)). Animals were then sacrificed 21 wk after the administration of the AAV9-Tert and AAV9-empty virus particles.
3
Propagation and Vaccination of BoDV-1 Virus
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After propagation of BoDV-1 virus “Dessau” in Newborn Rabbit Brain (NRB, permanent cell line established from the brains of newborn rabbits at Impfstoffwerk Dessau-Tornau, Dessau-Roßlau, Germany) cells (NRB/BDV), OL cells (OL/BDV cells), RK13 cells (RK13/BDV), MDBK cells (MDBK/BDV) the latter were chosen for cultivation of this virus in roller bottles (Corning, NY, USA, 850 cm2) using DMEM as medium. After 24 h the medium was changed to DMEM with 5% FBS supplemented with 10 mM n-butyrate. After 48 h, the medium was changed. to DMEM containing 300 mM NaCl. Two hours later the supernatant was taken and the virus content was determined. Proportions of the virus harvest were ultracentrifuged at 100,000× g for 120 min. The pellets were resuspended in 1 mL PBS (pH 7.2), pooled and further diluted with PBS. The virus content was adjusted to 107.1 FFU/mL. From the suspension two different vaccines were produced by mixing the supernatant with either 20% MontanideTM ISA25 (Seppic GmbH, Köln, Germany) (ISA25) or 20% PBS in order to generate one adjuvanted and one non-adjuvanted vaccine with same content of antigen (107 FFU/1 mL). The experimental vaccines were used for vaccination experiments in horses (dose 1 mL) for determination of antibody levels.
4
Generation and Purification of AAV Viral Vectors
5
Cultivation and Enumeration of Pathogenic Bacteria
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Mycobacterium tuberculosis H37Rv (ATCC 27294) was grown in suspension with constant gentle rotation in roller bottles (Corning, Corning, NY, United States) containing Middlebrook 7H9 broth (BD Biosciences, Franklin Lakes, NK, United States) supplemented with 1% glycerol (Roth, Karlsruhe, Germany), 0.05% Tween 80 (Sigma-Aldrich, Steinheim, Germany), and 10% Middlebrook oleic acid, albumin, dextrose, and catalase enrichment (BD Biosciences, OADC). Aliquots from logarithmically growing cultures were frozen at −80°C in 7H9 broth with 20% glycerol, and representative vials were thawed and enumerated for viable colony forming units (CFU) on Middlebrook 7H11 plates (BD Biosciences). Staining of bacterial suspensions with fluorochromic substrates differentiating between live and dead bacteria (BacLight, Invitrogen, Carlsbad, CA, United States) revealed a viability of the bacteria >90%. Thawed aliquots were sonicated in a water bath for 10 min at 40 kHz and 110 W before use. Pseudomonas aeruginosa (ATCC 27853), Extended Spectrum Beta-Lactamase (ESBL) Klebsiella pneumoniae (ATCC 7000603), and Escherichia coli DH5α (Law and Kelly, 1995 (link)) were cultured in liquid THY broth (Oxoid, ThermoFisher Scientific, Schwerte, Germany) supplemented with 0.5% yeast extract (BD Biosciences) at 37°C overnight in a 5% CO2 atmosphere.
6
Cultivation of Mycobacteria for Dormancy Studies
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Frozen aliquots of mycobacteria were thawed at room temperature and passed through a syringe for single cell separation. 200 µL bacterial culture was added to 5 mL Middlebrook 7H9 broth (Difco, Detroit, MI, USA) containing 0.2% glycerol, 0.05% Tween 80, and 10% oleic acid-albumin-dextrose-catalase (OADC) growth enrichment (Becton Dickinson, Cockeysville, MD, USA). Cultures were incubated at 37 °C until they reached an OD of ~ 0.2. Two mL pre-culture (at OD = 0.2) were added to 15–18 mL Dubos medium (Becton, Dickinson) supplemented with 0.05% Tween 80 and 10% OADC and incubated in roller bottles (Corning) at 37 °C for 7–10 days. At an OD of 0.4–0.5 (log phase) the cultures were diluted to OD = 0.004 in Dubos complete medium and used for dormancy experiments. Colony forming unit (CFU) analyses were calculated by serial dilutions plated on Middlebrook 7H11 agar containing 0.5% glycerol and 10% OADC growth enrichment. All CFU values in this study represent CFU/mL culture. A list of all used clinical isolates can be found in Supplementary Table S3 .
7
Cultivation and Enumeration of Mycobacterium tuberculosis
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Mycobacterium tuberculosis H37Rv (ATCC 27294, Manassas, VA, USA) was grown in suspension with constant gentle rotation in roller bottles (Corning, Corning, NY, USA) containing Middlebrook 7H9 broth (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 1% glycerol (Roth, Karlsruhe, Germany), 0.05% Tween 80 (Sigma-Aldrich, Steinheim, Germany), and 10% Middlebrook oleic acid, albumin, dextrose, and catalase enrichment (BD Biosciences, OADC) adjusted to pH 7.2–7.4. Aliquots from logarithmically growing cultures were kept frozen at −80 °C in 7H9 broth with 20% glycerol, and representative vials were thawed and enumerated for viable CFU on Middlebrook 7H11 plates (BD Biosciences, Franklin Lakes, NJ, USA). Staining of bacterial suspensions with fluorochromic substrates differentiating between live and dead bacteria (BacLight, Invitrogen, Carlsbad, CA, USA) revealed a viability of the bacteria >90% and confirmed the absence of large bacterial aggregates. Thawed bacterial stock aliquots were sonicated for 10 min at 40 kHz and 110 W in a Transsonic digital S (Elma, Wetzikon, Switzerland) before use.
8
Optimized Production and Purification of AAV9 Viral Vectors
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Viral vectors were generated as described (Matsushita et al., 1998 (link)) and purified as previously described (Ayuso et al., 2014 (link)). Vectors were produced through triple transfection of HEK293T. Cells were grown in roller bottles (Corning, NY, USA) in DMEM medium supplemented with fetal bovine serum (10% v/v) to 80% confluence and then co-transfected with the following plasmids: plasmid_1 carrying the expression cassette for gene of interest flanked by the AAV2 viral ITRs; plasmid_2 carrying the AAV rep2 and cap9 genes; plasmid_3 carrying the adenovirus helper functions (plasmids were kindly provided by K.A. High, Children’s Hospital of Philadelphia). The expression cassettes were under the control of the cytomegalovirus (CMV) promoter and contained a SV40 polyA signal for EGFP and the CMV promoter and the 3’-untranslated region of the Tert gene as polyA signal for Tert. AAV9 particles were purified following an optimized method using two caesium chloride gradients, dialysed against PBS, filtered and stored at 80°C until use. Mice were injected via tail vein IV with 100 μL of rAVV9 viral genome particle (2.5*1013 vg/mL).
9
Monoclonal Antibody Isotyping and Production
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Identification of antibody subclasses was performed using a Mouse Monoclonal Antibody Isotyping Kit (ISO2, Sigma). For large-scale mAb production hybridoma cell lines were cultured in 500 ml roller-bottles (Corning). Monoclonal antibodies were purified by affinity chromatography using protein A sepharose (GE Healthcare).
10
Mycobacterial Growth Conditions Protocol
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Mtb H37Rv and Msmeg mc2 155 were cultured in 7H9 broth or on 7H10 plates (Difco Laboratories), supplemented with 0.5% glycerol and 10% ADS (50 g/L Bovine Albumin Fraction V, 20 g/L D-glucose, 8.1 g/L NaCl). To test growth on single nitrogen sources, bacteria were grown in minimal medium (10% basic salts, 0.2% trace elements, 0.5 mM MgCl2, 0.5 mM CaCl2), supplemented with 0.5% glycerol, 10% ADS, and 5 mM of the indicated nitrogen source. Basic salts combined 10 g/L KH2PO4, 25 g/L Na2HPO4 and 20 g/L K2SO4. The trace elements solution contained 40 mg/L of ZnCl2, 200 mg/L of FeCl3, 10 mg/L of CuCl2, 10 mg/L of MnCl2, and 10 mg/L of Na2B4O7. Growth in presence of single carbon sources was performed in Sauton’s modified medium [39 (link)] supplemented with the indicated concentrations of arginine or glycerol. For all liquid media, 0.05% tyloxapol was added. Mycobacteria were incubated in roller bottles (Corning) at 70 rpm (Innova 4000 Benchtop Incubator Shaker, New Brunswick Scientific).
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