dnase, by Serva Electrophoresis - Product details

1

Isolation of Mouse Lung Cell Types

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Mouse lungs were perfused with HBSS (Gibco) followed by instillation of dispase
(BD Biosciences) into the lung through the trachea and incubation in dispase for
40 minutes as previously described56 (link). Trachea and
large airways were dissected and the remaining distal lung tissue was
homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01%
DNase (Serva) and filtered through 100 μm and
40 μm nylon filters. Cell suspensions were incubated
with biotinylated rat anti-mouse CD45, CD16/32 and CD31 mAb (BD
Biosciences) for 30 minutes at 37 °C
followed by incubation with biotin-binding magnetic beads and magnetic
separation to deplete leukocytes and endothelial cells prior to flow cytometric
analysis.

Reuther P., Göpfert K., Dudek A.H., Heiner M., Herold S, & Schwemmle M. (2015). Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment. Scientific Reports, 5, 11346.

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2

Isolation and Culture of Alveolar Epithelial Cells

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Lung homogenates were obtained by instillation of dispase (BD Biosciences) through the trachea into HBSS (Gibco) perfused lungs, followed by incubation (in dispase) for 40 min as previously described^{61 (link)}. After removal of the trachea and proximal bronchial tree, the lungs were homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01% DNase (Serva) and filtered through 100 and 40 μm nylon filters. Cell suspensions were incubated with biotinylated rat anti-mouse CD45 (Clone 30-F11, BD, Cat No 553078), CD16/32 (clone 2.4G2, BD, Cat No 553143) and CD31 (clone MEC13.3, BD, Cat No 553371) mAbs for 30 min at 37 °C followed by incubation with biotin-binding magnetic beads and magnetic separation to deplete leukocytes and endothelial cells prior to further culture. AEC suspensions with a purity ≥90% as determined by FACS were seeded at a density of 120–150,000 cells/cm² in 4-μm–pore size transwells (Corning Inc.), in 24-well plates at a density of 250,000 cells/cm², or in chamber slides (Corning Inc.), and cultured in DMEM enriched with HEPES, L-Glutamine, FCS, and pen/strep. The list of antibodies in Excel format including clone, company name, Cat No is provided as Supplementary Data 1.

Pervizaj-Oruqaj L., Selvakumar B., Ferrero M.R., Heiner M., Malainou C., Glaser R.D., Wilhelm J., Bartkuhn M., Weiss A., Alexopoulos I., Witte B., Gattenlöhner S., Vadász I., Morty R.E., Seeger W., Schermuly R.T., Vazquez-Armendariz A.I, & Herold S. (2024). Alveolar macrophage-expressed Plet1 is a driver of lung epithelial repair after viral pneumonia. Nature Communications, 15, 87.

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3

P2ry2 Expression in Lung Cells

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Resected lungs from P2ry2^fl/fl Cct-cre⁺ mice and cre-negative littermates were filled up with 1 mL dispase (Corning Incorporated, USA) and digested in Dulbecco’s Modified Eagle Medium (DMEM) containing 2 µg/mL collagenase (Sigma-Aldrich, Germany) and 0.001% DNAse (Serva, Germany). Anti-mouse CD31⁻ (Biolegend, CA, USA), anti-mouse CD45⁻ (Biolegend, CA, USA), and anti-mouse EpCAM⁺ (Thermo Scientific, Germany) cells were sorted with FACS Aria III (BD Biosciences, Germany). Subsequent qPCR was performed to evaluate P2ry2 expression.

Schneble D., El-Gazzar A., Kargarpour Z., Kramer M., Metekol S., Stoshikj S, & Idzko M. (2023). Cell-type-specific role of P2Y2 receptor in HDM-driven model of allergic airway inflammation. Frontiers in Immunology, 14, 1209097.

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4

Lung Cell Isolation and FACS Analysis

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The lungs were isolated and filled up with 2 ml dispase (cat# 354235; BD Bioscience). After mincing with a sterile scalpel, the lung tissue was incubated with 2 μg/ml collagenase B (cat# 11088815001; Life Sciences, Darmstadt, Germany) and 0.001% DNAse (cat#18535; Serva Electrophoresis, Heidelberg, Germany) in DMEM for 15 min. at 37°C. The resulting cell suspension was filtered through a 100 μm and 40 μm cell strainers (cat# 352340; BD Falcon, Heidelberg, Germany), harvested by centrifugation and resuspended in cell blood lysis buffer (0.15 mol NH₄Cl, 10 mmol KHCO₃, 0.1 mmol EDTA). After washes in PBS, the cells were resuspended in FACS-buffer (PBS, 2 mmol EDTA, 25 mmol HEPES) at 1 million cells per 100 μl buffer and incubated for 20 min. on ice with a mixture of anti-EpCAM and anti-CD45 antibodies (e-Biosciences, Frankfurt, Germany). The cells were analysed with a BD LSR II flow cytometer by using flowJo7.6.4 software, or were sorted with a BD FACSAria III cell sorter.

Galiger C., Kostin S., Golec A., Ahlbrecht K., Becker S., Gherghiceanu M., Popescu L.M., Morty R.E., Seeger W, & Voswinckel R. (2014). Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung. Journal of Cellular and Molecular Medicine, 18(7), 1321-1333.

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5

Isolation and Purification of Bacterial O-Antigens

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The LPS was isolated applying the hot phenol-water method⁵⁵, followed by dialysis against distilled water until the phenol scent was gone. Then samples were treated with DNase (1mg/100 mg LPS) plus RNase (2 mg/100 mg LPS) at 37°C for 2 h, followed by Proteinase K treatment (1 mg/100 mg LPS) at 60°C for 1 h [all enzymes from Serva, Germany]. Subsequently, samples were dialyzed again for 2 more days, then freeze dried. Such LPS samples were then hydrolyzed with 1% aqueous acetic acid (100°C, 90 min) and ultra-centrifuged for 16 h at 4°C and 150,000 g. Resulting supernatants (the O-antigens) were dissolved in water and freeze-dried. For further purification, the crude O-antigen samples were chromatographed on TSK HW-40 eluted with pyridine/acetic acid/water (10/4/1000, by vol.), then lyophilized. On these samples, 1D and 2 D (COSY, TOCSY, HSQC, HMBC) ¹H- and ¹³C-NMR spectra were recorded with a Bruker DRX Avance 700 MHz spectrometer (¹H: 700.75 MHz; ¹³C: 176.2 MHz) as described^{56 (link)}.

Diard M., Bakkeren E., Lentsch V., Rocker A., Bekele N.A., Hoces D., Aslani S., Arnoldini M., Böhi F., Schumann-Moor K., Adamcik J., Piccoli L., Lanzavecchia A., Stadtmueller B.M., Donohue N., van der Woude M.W., Hockenberry A., Viollier P.H., Falquet L., Wüthrich D., Bonfiglio F., Loverdo C., Egli A., Zandomeneghi G., Mezzenga R., Holst O., Meier B.H., Hardt W.D, & Slack E. (2021). A rationally designed oral vaccine induces Immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants. Nature microbiology, 6(7), 830-841.

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6

Isolation of Distal Lung Cell Suspensions

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Lung homogenates of distal lung cell suspensions were obtained by instillation of dispase (BD Biosciences) and 0.5 ml low-melting agarose (Sigma) through the trachea into the HBSS (Gibco) perfused lung, followed by incubation in dispase for 40 min as previously described [55 (link)]. After gelling of the agarose and removal of the agarose-filled trachea and proximal bronchial tree, the lung was homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01% DNase (Serva) and filtered through 100μm and 40μm nylon filters. Cell suspensions were incubated with biotinylated rat anti-mouse CD45, CD16/32 and CD31 mAb for 30 min at 37°C followed by incubation with biotin-binding magnetic beads and magnetic separation to deplete leukocytes and endothelial cells prior to flow cytometric analysis and cell sorting or to further culture.

Quantius J., Schmoldt C., Vazquez-Armendariz A.I., Becker C., El Agha E., Wilhelm J., Morty R.E., Vadász I., Mayer K., Gattenloehner S., Fink L., Matrosovich M., Li X., Seeger W., Lohmeyer J., Bellusci S, & Herold S. (2016). Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair. PLoS Pathogens, 12(6), e1005544.

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7

Isolation and Purification of Bacterial O-Antigens

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The LPS was isolated applying the hot phenol-water method⁵⁵, followed by dialysis against distilled water until the phenol scent was gone. Then samples were treated with DNase (1mg/100 mg LPS) plus RNase (2 mg/100 mg LPS) at 37°C for 2 h, followed by Proteinase K treatment (1 mg/100 mg LPS) at 60°C for 1 h [all enzymes from Serva, Germany]. Subsequently, samples were dialyzed again for 2 more days, then freeze dried. Such LPS samples were then hydrolyzed with 1% aqueous acetic acid (100°C, 90 min) and ultra-centrifuged for 16 h at 4°C and 150,000 g. Resulting supernatants (the O-antigens) were dissolved in water and freeze-dried. For further purification, the crude O-antigen samples were chromatographed on TSK HW-40 eluted with pyridine/acetic acid/water (10/4/1000, by vol.), then lyophilized. On these samples, 1D and 2 D (COSY, TOCSY, HSQC, HMBC) ¹H- and ¹³C-NMR spectra were recorded with a Bruker DRX Avance 700 MHz spectrometer (¹H: 700.75 MHz; ¹³C: 176.2 MHz) as described^{56 (link)}.

Diard M., Bakkeren E., Lentsch V., Rocker A., Bekele N.A., Hoces D., Aslani S., Arnoldini M., Böhi F., Schumann-Moor K., Adamcik J., Piccoli L., Lanzavecchia A., Stadtmueller B.M., Donohue N., van der Woude M.W., Hockenberry A., Viollier P.H., Falquet L., Wüthrich D., Bonfiglio F., Loverdo C., Egli A., Zandomeneghi G., Mezzenga R., Holst O., Meier B.H., Hardt W.D, & Slack E. (2021). A rationally designed oral vaccine induces Immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants. Nature microbiology, 6(7), 830-841.

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8

Purification of His-Tagged Recombinant Proteins

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Escherichia coli cells were grown in LB medium with appropriate antibiotics at 220 rpm and 37 °C to an A600 of 0.6. Protein expression in E. coli BL21 was induced by 1 mM IPTG for two hours at 30 °C. His-tagged PSK1 or RGF1 precursors were purified from bacterial extracts by metal chelate affinity chromatography on NiNTA Agarose (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Cells were harvested by centrifugation (4000 x g, 4 °C, 20 min) and resuspended in 50 mM sodium phosphate buffer, pH 7.0, supplemented with 300 mM NaCl including lysozyme, PMSF, DNAse and Proteinase Inhibitor Cocktail (Serva). Cells were lysed by sonication (SONOPULS HB2070 with MS72 sonic needle, Bandelin Electronics; Berlin, Germany) thrice at 4 °C for 30 seconds. The soluble fraction was collected by centrifugation (15000 x g, 4 °C, 10 min).
Recombinant proteins were bound to the Ni-NTA column for at least one hour. The column was washed extensively in buffer containing 10 mM imidazole and recombinant proteins were eluted in buffer containing 200 mM imidazole. After ultrafiltration (30 kDa molecular mass cutoff), the recombinant protein in the filtrate was further purified by size exclusion chromatography (NGC TM chromatography system with Enrich TM SEC 650 column, BioRad, Munich, Germany) in 50 mM NaH2PO4 /Na2HPO4, pH 5.5 (or 7.0 if indicated), 10 mM NaCl.

Stührwohldt N., Bühler E., Sauter M, & Schaller A. (2021). Phytosulfokine (PSK) precursor processing by subtilase SBT3.8 and PSK signaling improve drought stress tolerance in Arabidopsis. Journal of experimental botany, 72(9).

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9

Purification of His-Tagged Recombinant Proteins

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Escherichia coli cells were grown in LB medium with appropriate antibiotics at 220 rpm and 37 °C to an A600 of 0.6. Protein expression in E. coli BL21 was induced by 1 mM IPTG for two hours at 30 °C. His-tagged PSK1 or RGF1 precursors were purified from bacterial extracts by metal chelate affinity chromatography on NiNTA Agarose (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Cells were harvested by centrifugation (4000 x g, 4 °C, 20 min) and resuspended in 50 mM sodium phosphate buffer, pH 7.0, supplemented with 300 mM NaCl including lysozyme, PMSF, DNAse and Proteinase Inhibitor Cocktail (Serva). Cells were lysed by sonication (SONOPULS HB2070 with MS72 sonic needle, Bandelin Electronics; Berlin, Germany) thrice at 4 °C for 30 seconds. The soluble fraction was collected by centrifugation (15000 x g, 4 °C, 10 min).
Recombinant proteins were bound to the Ni-NTA column for at least one hour. The column was washed extensively in buffer containing 10 mM imidazole and recombinant proteins were eluted in buffer containing 200 mM imidazole. After ultrafiltration (30 kDa molecular mass cutoff), the recombinant protein in the filtrate was further purified by size exclusion chromatography (NGC TM chromatography system with Enrich TM SEC 650 column, BioRad, Munich, Germany) in 50 mM NaH2PO4 /Na2HPO4, pH 5.5 (or 7.0 if indicated), 10 mM NaCl.

Stührwohldt N., Bühler E., Sauter M., & Schaller A. (2020). Precursor processing by SBT3.8 and phytosulfokine signaling contribute to drought stress tolerance in Arabidopsis.

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Dnase

Lab products found in correlation

9 protocols using dnase

Isolation of Mouse Lung Cell Types

Isolation and Culture of Alveolar Epithelial Cells

P2ry2 Expression in Lung Cells

Lung Cell Isolation and FACS Analysis

Isolation and Purification of Bacterial O-Antigens

Isolation of Distal Lung Cell Suspensions

Isolation and Purification of Bacterial O-Antigens

Purification of His-Tagged Recombinant Proteins

Purification of His-Tagged Recombinant Proteins

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