Two nasal swabs (one per nostril) were taken daily following infection with wt H1N1 and immunization with LAIV and following subsequent challenge with wt H1N1. The swabs were placed into 2 ml of virus transport medium comprising tissue culture medium 199 (Sigma-Aldrich, UK) supplemented with 25 mM Hepes, 0.035% sodium bicarbonate, 0.5% BSA, penicillin, streptomycin and nystatin, vortexed, centrifuged to remove debris and stored at −80°C for subsequent virus titration. Serum and heparin anti-coagulated blood samples were collected at the start of the study (prior to LAIV immunization or wt H1N1 pre-exposure), before challenge and four dpc at post-mortem. Heparin blood samples were diluted 1:1 in PBS before density gradient centrifugation at 1,200 × g for 30 min over Histopaque® 1.083 g/ml (Sigma-Aldrich, UK). PBMC were harvested from the interface, washed and red blood cells lysed with Red Blood Cell Lysis Buffer (BioLegend, UK), washed again and cryopreserved in FCS (Gibco) with 10% DMSO (Sigma-Aldrich, UK). Broncho-alveolar lavage (BAL) and tracheobronchial lymph nodes (TBLN) were processed as previously described (38 (link)).
Tissue culture medium 199
Manufactured by Merck Group
Sourced in United Kingdom, United States
Tissue culture medium 199 is a commonly used cell culture medium that provides a balanced combination of nutrients and growth factors to support the in vitro cultivation of a variety of cell types, including mammalian, avian, and insect cells. It is a complex, buffered medium that contains a variety of amino acids, vitamins, salts, and other essential components required for cell growth and maintenance.
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7 protocols using tissue culture medium 199
1
Immune response assessment following influenza exposure
2
Intranasal H1N1pdm09 Viral Challenge
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Two nasal swabs (one per nostril) were taken each day following the challenge with H1N1pdm09. The swabs were placed into 2 ml of virus transport medium (VTM) comprising tissue culture medium 199 (Sigma-Aldrich) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.035% sodium bicarbonate, 0.5% bovine serum albumin, penicillin 100 IU/ml, streptomycin 100 µg/ml, and nystatin 0.25 µg/ml, vortexed, centrifuged to remove debris, aliquoted and stored at −80°C for subsequent virus titration. Serum samples were collected at the start of the study (prior to mAb administration and challenge) and at 0, 1, 3, and 4 DPI of the challenge. Broncho-alveolar lavage fluid (BAL) was collected from the entire left lung with 100 ml of 0.1% BSA+PBS. BAL samples were centrifuged at 300×g for 15 min, supernatant was removed, aliquoted, and frozen for antibody and virus titer analysis. The accessory lung lobes were dissected out and frozen at −80°C for subsequent virus titration. The whole lobe was cut into pieces and 10% (w/v) pieces of the lung were homogenized in 0.1% BSA using the gentle MACS Octo dissociator, the homogenate was clarified by centrifugation and the supernatant was used for virus titration.
3
Multisite Sampling for SARS-CoV-2 Detection
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Four nasal swabs (NS) (two per nostril) were taken at 0, 1, 2, 3, and 4 dpi. The swabs were placed into 2 ml of virus transport medium comprising tissue culture medium 199 (Sigma-Aldrich) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.035% sodium bicarbonate, 0.5% bovine serum albumin, penicillin 100 IU/ml, streptomycin 100 µg/ml, and nystatin 0.25 µg/ml, vortexed, centrifuged to remove debris, and stored at −80oC for subsequent virus titration. Serum samples were collected at the start of the study (prior to challenge) and at 2 and 4 dpi. For Fc binding and ADCC assays, blood from healthy humans and uninfected pigs was used. Heparinized blood samples were diluted 1:1 in PBS before density gradient centrifugation. PBMC were harvested from the interface, washed and red blood cells lysed with ammonium chloride lysis buffer, washed again, and used in Fc binding and ADCC assays described below. Broncho-alveolar lavage (BAL) was collected from the entire left lung with 150 ml of virus transport medium (described above). BAL samples were centrifuged at 300 × g for 15 min, supernatant was removed, aliquoted, and frozen for antibody analysis.
4
Nasal Swabs and Biofluid Sampling
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Four nasal swabs (two per nostril) were taken at 0, 1, 2, 3, 4 DPI. The swabs were placed into 1 ml of TRIzol or 2 ml of virus transport medium comprising tissue culture medium 199 (Sigma-Aldrich, St. Louis, MO) supplemented with 25 mM HEPES, 0.035% sodium bicarbonate, 0.5% BSA, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml nystatin, vortexed, centrifuged to remove debris, and stored at −80°C for subsequent virus titration. Blood samples were collected at the start of the study (prior to Ab administration) and at the indicated times post-mAb delivery and challenge. BAL was collected from the entire left lung with 150 ml of virus transport medium (described above). BAL samples were centrifuged at 300 × g for 15 min and the supernatant was removed, aliquoted, and frozen for Ab analysis.
5
Rat Platelet Isolation and Preparation
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Blood was drawn from the rats 20 hours after the last injection of S1R ligands had been administered. Under anesthesia (Euthasol®/pentobarbital-Na/ 30 mg/kg body weight i.p.), blood was drawn from the abdominal aorta of rats with a thick needle and diluted (1:2) with phosphate buffer (pH 7.4) containing ethylenediaminetetraacetic acid (EDTA, 5.8 mM) and glucose (5.55 mM). The platelets were separated by differential centrifugation [29 (link), 30 (link)]. The platelet-rich plasma was collected after the whole blood had been centrifuged at 200 g for 10 min at room temperature. The platelets were sedimented from the supernatant by centrifugation at 2000 g for 10 min. The pellet was contaminated with red blood cells; the erythrocytes were therefore lysed with hypoosmotic ammonium chloride (0.83%, 9 parts) containing EDTA (0.02%, 1 part) over 15 min. The platelets were then washed twice with phosphate buffer pH 7.4 containing 5.8 mM EDTA and 5.55 mM glucose and centrifuged at 2000 g for 10 min at room temperature to remove the ammonium chloride and the erythrocyte residue/debris. The absolute platelet count was determined before the second centrifugation. After the last centrifugation, the platelets were re-suspended (2.5 x 108 platelets/mL) in serum-free Medium 199 tissue culture (Sigma, St. Louis, MO, USA) [19 (link)].
6
Rat Platelet Isolation and Purification
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Blood was drawn with a thick needle from the abdominal aorta of the rats under anasthesia (Euthasol ® /pentobarbital-Na/ 30 mg/kg body weight i.p.) and diluted (1:2) with a phosphate buffer (pH 7.4) containing ethylene diamino tetraacetic acid (EDTA, 5.8 mM) and glucose (5.55 mM). Platelets were separated by differential centrifugation (Mezei et al., 1997) (link).
Platelet-rich plasma was collected after the whole blood had been centrifuged at 200 g, for 10 min, at room temperature. Platelets were sedimented from the supernatant by centrifugation at 2000 g, for 10 min. The pellet was contaminated with red blood cells; erythrocytes were therefore lysed for over 15 min with hyposmotic ammonium chloride (0.83%, 9 parts) containing EDTA (0.02%, 1 part). The platelets were then washed twice with phosphate buffer pH 7.4, containing 5.8 mM EDTA and 5.55 mM glucose, and centrifuged at 2000 g, for 10 min, at room temperature to remove the ammonium chloride and the erythrocyte residues/debris. The absolute platelet count was determined before the second centrifugation. After the last centrifugation, the platelets were resuspended (2.5x10 8 platelets/mL) in serum-free Medium 199 tissue culture (Sigma, St. Louis, MO).
Platelet-rich plasma was collected after the whole blood had been centrifuged at 200 g, for 10 min, at room temperature. Platelets were sedimented from the supernatant by centrifugation at 2000 g, for 10 min. The pellet was contaminated with red blood cells; erythrocytes were therefore lysed for over 15 min with hyposmotic ammonium chloride (0.83%, 9 parts) containing EDTA (0.02%, 1 part). The platelets were then washed twice with phosphate buffer pH 7.4, containing 5.8 mM EDTA and 5.55 mM glucose, and centrifuged at 2000 g, for 10 min, at room temperature to remove the ammonium chloride and the erythrocyte residues/debris. The absolute platelet count was determined before the second centrifugation. After the last centrifugation, the platelets were resuspended (2.5x10 8 platelets/mL) in serum-free Medium 199 tissue culture (Sigma, St. Louis, MO).
7
Rat Platelet Isolation Using Differential Centrifugation
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Blood was drawn from the rats 20 hours after the last injection of S1R ligands had been received. Under anesthesia (Euthasol®/pentobarbital-Na/ 30 mg/bwkg i.p.), blood was drawn from the abdominal aorta of rats with a thick needle and was diluted (1:2) with phosphate buffer (pH 7.4) containing ethylene diamine tetra acetic acid (EDTA, 5.8 mM) and glucose (5.55 mM).
Platelets were separated by differential centrifugation. 24, 25 Platelet-rich plasma was collected after the whole blood had been centrifuged at 200 g, for 10 min, at room temperature. Platelets were sedimented from the supernatant by centrifugation at 2000 g, for 10 min. The pellet was contaminated with red blood cells; therefore, erythrocytes were lysed with hypoosmotic ammonium chloride (0.83%, 9 parts) containing EDTA (0.02%, 1 part) over 15 min. The platelets were then washed twice with phosphate buffer pH 7.4 containing 5.8 mM EDTA and 5.55 mM glucose, and centrifuged at 2000 g, for 10 min, at room temperature to remove the ammonium chloride and the erythrocyte residues/debris. The absolute platelet count was determined before the second centrifugation. After the last centrifugation, the platelets were resuspended (2.5x10 8 platelets/mL) in serum-free Medium 199 tissue culture (Sigma, St. Louis, MO).
Platelets were separated by differential centrifugation. 24, 25 Platelet-rich plasma was collected after the whole blood had been centrifuged at 200 g, for 10 min, at room temperature. Platelets were sedimented from the supernatant by centrifugation at 2000 g, for 10 min. The pellet was contaminated with red blood cells; therefore, erythrocytes were lysed with hypoosmotic ammonium chloride (0.83%, 9 parts) containing EDTA (0.02%, 1 part) over 15 min. The platelets were then washed twice with phosphate buffer pH 7.4 containing 5.8 mM EDTA and 5.55 mM glucose, and centrifuged at 2000 g, for 10 min, at room temperature to remove the ammonium chloride and the erythrocyte residues/debris. The absolute platelet count was determined before the second centrifugation. After the last centrifugation, the platelets were resuspended (2.5x10 8 platelets/mL) in serum-free Medium 199 tissue culture (Sigma, St. Louis, MO).
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