2,5-Dihydroxybenzoic acid (DHB), norharmane, Fluoroshield with DAPI, Monoclonal Anti-Actin α-Smooth Muscle antibody, LC-MS grade methanol, HPLC grade chloroform, Bouin’s solution, Triton X-100, Tween 20, poly-L-lysine solution 0.1% and Peel-a-Way embedding molds (truncated, 12 × 12 × 20 mm) were purchased from Sigma-Aldrich, Saint Louis, MO, USA. Histology-grade solvents, Eosin G or Y (alcoholic solution 0.5%) and Masson’s Trichrome staining kit were purchased from DiaPath S.p.A., Martinengo, Italy, while QA-Agarose low melting point was obtained from MP Biomedical, Santa Ana, CA, USA. Harris hematoxylin, phosphate buffer saline (PBS) tablets, bovine serum albumin (BSA), SuperFrost Plus glass slides and cover slides were purchased from VWR International, LLC, Radnor, PA, USA. Trifluoroacetic acid (TFA), Antihuman CD68 antibody and AlexaFluor 568 goat anti mouse secondary antibody were purchased from Life Technologies-Invitrogen, Carlsbad, CA, USA. LC-MS grade water was purchased from Thermo Fisher Scientific, Rockford, IL, USA, while ITO coated glass slides for MALDI were obtained from Bruker Daltonics, Billerica, MA, USA. Paraformaldehyde 4% solution was purchased from Alfa Aesar, Haverhill, MA, USA.
Poly l lysine solution 0.1
Manufactured by Merck Group
Sourced in United States
Poly-L-lysine solution 0.1% is a laboratory product manufactured by Merck Group. It is a water-based solution containing poly-L-lysine at a concentration of 0.1% (w/v).
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Cl amidine, by Merck Group (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 568 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hplc grade chloroform, by Merck Group (1 mentions)
Lc ms grade water, by Thermo Fisher Scientific (1 mentions)
Superfrost plus glass slide, by Avantor (1 mentions)
Tween 20, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Fluoroshield with dapi, by Merck Group (1 mentions)
Bouin s solution, by Merck Group (1 mentions)
Harris hematoxylin, by Avantor (1 mentions)
2 5 dihydroxybenzoic acid, by Merck Group (1 mentions)
Norharmane, by Merck Group (1 mentions)
Lc ms grade methanol, by Merck Group (1 mentions)
Peel a way embedding molds, by Merck Group (1 mentions)
Antihuman cd68 antibody, by Thermo Fisher Scientific (1 mentions)
Camostat, by Merck Group (1 mentions)
Wright giemsa solution, by Muto Pure Chemicals (1 mentions)
6 protocols using poly l lysine solution 0.1
1
Comprehensive Tissue Preparation Protocol
2
Airway Fluid Sampling for COVID-19 NET Analysis
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As previously described [7 (link)], the airway fluid from 11 patients with COVID-19 patients was obtained by aspiration of the orotracheal tube. This fluid was mixed 1:1 with 0.1 M dithiothreitol (Thermo Fisher Scientific; cat. R0862), incubated for 15 min stirring every 5 min at 37 °C. In the control group (n=8), the airway lavage was obtained by injecting sterile isotonic saline solution through a nasal fossa. The injected solution was mixed with nasal and nasopharyngeal secretions before being evacuated from the other nostril when it was collected directly in a sterile tube. The samples were centrifuged at 750 g at 4 °C for 10 min. The supernatants were used for measurement of NETs, and the cells were fixed for immunostaining in coverslips with Poly-L-lysine solution 0.1% (Sigma-Aldrich; cat P8920).
3
Neutrophil Response to SARS-CoV-2 Infection
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Neutrophils (106 cells) obtained from COVID-19 patients or healthy controls were incubated with RPMI 1640 supplemented with 0.1% BSA treated or not with Cl-Amidine (200 µM; Sigma-Aldrich; cat. 506282), tenofovir disoproxil fumarate (TDF; 10 µM; as described in Clososki et al., 2020 (link)), neutralizing anti-ACE2 antibody (αACE2; 0.5 µg/ml; Rhea Biotech; cat. IM-0060), Camostat mesylate (Camostat; 10 µM; Sigma-Aldrich; cat. SML0057). All compounds were used 1 h before infection with SARS-CoV-2 (MOI = 1.0) or stimulation with 50 nM PMA. The viral load in cell pellet and concentration of NETs in supernatants was determined 4 h after infection. In another context, neutrophils from healthy controls were incubated with SARS-CoV-2 (MOI = 0.5 and 1.0) for 4 h at 37°C or inactivated SARS-CoV-2. The concentration of NETs in supernatants was determined. A total of 5 × 104 isolated neutrophils were attached to coverslips coated with poly-L-lysine solution 0.1% (Sigma-Aldrich; cat. P8920) incubated for 4 h at 37°C for NET immunostaining.
4
Staining and Mounting Nucleated Cells
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The NACs were centrifuged at 800 g for 10 min., washed with PBS, and then suspended in PBS. The smears of cell suspension were prepared on slide glass that was coated with poly-L-lysine solution 0.1% (Sigma-Aldrich, St. Louis, MO, USA). The slides were left to dry, fixed with methanol, and then stained with Wright–Giemsa solution (Muto Pure Chemicals, Japan).
5
Investigating SARS-CoV-2 Induced NETosis
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Neutrophils (1x10 6 cells) obtained from COVID-19 patients or healthy controls were incubated with RPMI 1640 supplemented with 0.1% BSA treated or not with Cl-Amidine (200 μM, Sigma-Aldrich, cat. 506282) for 4 h at 37°C. In another context, neutrophils from healthy controls were incubated with SARS-CoV-2 (MOI= 0.5 and 1.0) for 4 h at 37°C or inactivated SARS-CoV-2. The concentration of NETs in supernatants was determined. A total of 5x10 4 isolated neutrophils were attached to coverslips coated with poly-L-lysine solution 0.1% (Sigma-Aldrich, cat. P8920) incubated for 4 h at 37ºC for NETs immunostaining.
6
Airway Fluid Analysis of COVID-19 Patients
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This procedure was performed as previously described (Veras et al., 2020) (link). Briefly, the airway fluid from 11 patients with COVID-19 patients was obtained by aspiration of the . orotracheal tube. This fluid was mixed 1:1 with 0.1 M dithiothreitol (Thermo Fisher Scientific; cat. R0862), incubated for 15 min stirring every 5 min at 37°C. In the control group (n=8), the airway lavage was obtained by injecting sterile isotonic saline solution through a nasal fossa. The injected solution was mixed with nasal and nasopharyngeal secretions before being evacuated from the other nostril when it was collected directly in a sterile tube. The samples were centrifuged 750 g at 4°C for 10 min. The supernatants were used for measurement of NETs and the cells were fixed for immunostaining in coverslips with Poly-L-lysine solution 0.1% (Sigma-Aldrich; cat P8920).
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