Sputum collection and analysis were performed as previously described.22 Briefly, sputum induction was performed using inhalation of hypertonic saline (4.5%) through a mouthpiece connected to an ultrasonic nebulizer (OMRON NE-U17; Omron Healthcare Europe, Netherlands).23 (link) Sputum was then sampled and treated with dithiothreitol (Sputolysin; Calbiochem Corporation, USA). The suspension was centrifuged; the cell pellet was resuspended while the supernatant was collected and stored. Cytospins were prepared and stained using May-Grunwald/Giemsa. Differential cell counts were made by counting a minimum of 500 nonsquamous cells. The lower respiratory origin was confirmed by the presence of macrophages and bronchial epithelial cells. Normal values for sputum differential cell percentages were based on published healthy subjects counts.24 (link)25 (link)26 (link)27 (link) These measurements were done blindly to the clinical characteristics of the subjects.
Ne u17
Manufactured by Omron
Sourced in Japan, United Kingdom
The NE-U17 is a compact and portable ultrasonic nebulizer designed for laboratory use. It generates a fine mist from liquid solutions, enabling efficient aerosolization for various applications. The device operates using ultrasonic technology to produce a consistent particle size distribution.
Automatically generated - may contain errors
Lab products found in correlation
Methacholine, by Merck Group (2 mentions)
Dry bellows spirometer, by Vitalograph (1 mentions)
Kwik diff stain, by Thermo Fisher Scientific (1 mentions)
Balb c mice, by Samtako (1 mentions)
Methacholine, by Omron (1 mentions)
Aluminum hydroxide, by Merck Group (1 mentions)
Imject alum, by Thermo Fisher Scientific (1 mentions)
Ova grade 5, by Merck Group (1 mentions)
Ultrasonic nebulizer, by Omron (1 mentions)
17 protocols using ne u17
1
Sputum Collection and Analysis Protocol
2
Spirometry and Sputum Induction Protocol
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FEV1 was measured as the greater of two values within 100 ml using a dry bellows spirometer (Vitalograph, Buckingham, UK), prior to and 15 minutes after the inhalation of 400 μg of salbutamol via a spacer (VolumaticTM, Allen & Hanburys Ltd, Middlesex, UK). Sputum induction with hypertonic saline was performed as described previously [21 , 22 (link)] using the ultrasonic nebulizer OMRON NE-U17 (OMRON Health Care Ltd, Milton Keynes, UK). A clinician reviewed and examined participants at each visit.
3
Adoptive Transfer of Th9 Cells in Allergic Airway Inflammation
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Rag2−/− mice received 5 × 105 WT or Irf1−/− OTII-Th9 cells on day 0 by i.p. injection. Mice were then challenged via the airways with nebulized OVA (1% in saline) with an ultrasonic nebulizer (NE-U17; Omron, Hoofdrop, The Netherlands) for 20 min daily from day 1 to day 6. To neutralize IL-9 cytokine in vivo, mice were injected i.p. either with 30 μg anti-IL-9 (MM9C1, produced and purified ‘in house') or rat IgG (Sigma) antibody on days +1, +3 and +5. On day 7, BAL and lung tissue were collected.
4
AP-18 Aerosolization for Respiration
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AP-18 (ab144587, Abcam, Cambridge, England) was dissolved in 20% ethanol in saline. AP-18 was aerosolized with an ultrasonic nebulizer (NE-U17, Omron, Kyoto, Japan), mixed with air, and subsequently distributed into the whole-body plethysmography chamber. At the time of measurement, the supply of air and monitoring of O2 were paused for 3 min by closing the inlet and outlet stopcocks of the chamber to avoid fluctuation of the basal gas flow from vibration of the nebulizer because accurate recording of respiration by the flow-through type whole body plethysmography depends on constant air flow (Onodera et al., 1997 (link)).
5
Murine Model of Allergic Airway Inflammation
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Forty-nine female BALB/c mice were randomly divided into seven groups (seven mice per group) as follows: PBS administration (negative control), 10 mg/kg BKC administration, OVA sensitization and challenge, OVA sensitization and challenge with 0.1, 1, and 10 mg/kg BKC, and OVA sensitization and challenge with 10 mg/kg DEX. The mice were sensitized by intraperitoneal injection of an OVA mixture (100 µg OVA and 4 mg alum adjuvant in 200 µL PBS) on days 0, 7, and 14. On days 21 to 23, the mice were challenged by 3% OVA for 30 min. Finally, on day 24, the mice were challenged by 5% OVA. OVA exposure was conducted with an aerosol using an ultrasonic nebulizer (NE-U17; OMRON Corp., Tokyo, Japan). BKC or DEX was dissolved in 1% carboxymethyl cellulose and orally administered 1 h before the challenge. Negative control mice were given PBS instead of OVA in all procedures. After 24 h of the nal challenge, mice were euthanized with CO 2 , and blood was collected by cardiac puncture after collection of bronchoalveolar lavage uid (BALF). BALF was collected through 3 times of lung lavage with 0.5 mL autoclaved PBS using a tracheal cannula and then centrifuged at 400 × g for 10 min at 4°C. The supernatants of BALF were transfered to new micro tube and stored at -80°C until analysis. The pelleted cells were resuspended in 300 µL PBS and analyzed immediately.
6
Sputum Collection and Neutrophil Elastase Assay
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Sputum from patients with CF was obtained during a physiotherapy session while sputum from healthy subjects was induced by using the method of Pizzichini and colleagues, with minor modifications [26] (link). Sputum induction was achieved with an aerosol of hypertonic saline generated by the NE-U17 ultrasonic nebulizer (Omron). Healthy subjects equipped with a nose clip, inhaled increasing concentrations of saline (3, 4, and 5%) for 7 min each through a mouthpiece. We then asked subjects to rinse their mouth or swallow to minimise contamination with saliva. Finally, we invited them to cough sputum into a sterile container. Collected sputum was diluted 1:10 (w:v) in phosphate-buffered saline with 10 U/mL DNase (Pulmozyme 2500 U/2.5 mL – Roche, USA) and incubated under agitation, during 30 min, at 37°C. Then, it was filtered with 48 µm pore nylon filter (Prosep, Belgium) and centrifuged at 450 g, during 5 min. Neutrophil elastase activity was measured in supernatant. Remaining supernatant was treated with PMSF Protease Inhibitor 2% v:v in order to inactivate elastase and was stored at -80°C. Cells were processed for cytospin (fixed in methanol and staining by Diff-Quick method (Kwik-Diff™ Stains, Thermo Fisher Scientific) and epithelial contamination was assessed. The blood was centrifuged at 650 g for 20 min, the serum was collected, and stored at -80°C.
7
Ovalbumin-Induced Asthma Model in Mice
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Six-week-old female BALB/c mice (n=80; body weight, 22±2 g) were purchased from Samtako Bio Co. (Osan, Korea), fed with an ad libitum diet and water, and housed in a controlled environment (22±3°C, 12 h light/dark cycle). They were then divided into six treatment groups: (i) Control, (ii) sterilized tap water with ovalbumin (OVA) induction, (iii) 1 mg/kg/day dexamethasone (DEX; Sam Nam Pharm, Chungcheongnam, Korea) with OVA induction, (iv) 10 mg/kg/day M. atichisonii with OVA induction, (v) 100 mg/kg/day M. atichisonii with OVA induction and (vi) 1,000 mg/kg/day M. atichisonii with OVA induction. On days 1 and 8, all mice were sensitized via intraperitoneal injection of 20 µg OVA (cat no. A5503-50G; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 1 mg aluminum hydroxide hydrate (Prod #77161; Thermo Fisher Scientific, Inc. Waltham, MA, USA) in 500 µl saline. From days 21 to 25, all mice, except for those used as control, were challenged once daily with 5% OVA for 30 min using a nebulizer (3 ml/min, NE-U17; Omron Corporation, Kyoto, Japan). During the same 5 day period, the treatment groups were also administered once daily with oral doses of sterilized tap water, DEX, 10, 100 or 1,000 mg/kg/day M. atichisonii 1 h prior to the OVA challenge.
8
Noninvasive Airway Hyperresponsiveness Measurement
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Noninvasive AHR was measured using an OCP 3000 barometric plethysmographic chamber (Allmedicus, Anyang, Korea), as previously described.29 (link) Briefly, we prepared methacholine (Sigma-Aldrich) at the following concentrations: 6.25, 12.5, 25 and 50 mg/mL. Mice inhaled each concentration of methacholine for 3 minutes as an aerosol prepared with a NE-U17 (OMRON, Milton Keynes, UK), and enhanced pause (Penh) values were measured for 5 minutes. Airway resistance was expressed in Penh. Data are expressed as the percentage increase in Penh after challenge with each concentration of methacholine, where the baseline Penh (after PBS challenge) was set at 100%.
9
OVA-Induced Allergy Model in BALB/c Mice
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The BALB/c mice were randomly divided into five groups (n = 5) as follows: control, OVA, OVA + HPD 1 mg/kg (low-dose), OVA + HPD 10 mg/kg (high-dose), and OVA + Dex 2 mg/kg (positive control drug). The mice were intraperitoneally sensitized with the OVA mixture (OVA 100 μg + alum 4 mg in PBS 200 μL) on days 0 and 7. Then, the mice were challenged for 30 min with OVA 3% solution in PBS that was administered as an aerosol by using an ultrasonic nebulizer (NE-U17; OMRON Corp., Tokyo, Japan) on days 14–17. At the time of the challenge, the mice received daily oral administration of PBS, HPD, or Dex. On Day 18, all mice were euthanized via isoflurane inhalation, and blood, bronchoalveolar lavage fluid (BALF), lung, spleen, and lymph node samples were collected for analysis (Figure S1 ). The splenic weight was measured immediately after sacrifice; the BALF sample was centrifuged at 500× g for 5 min at 4 °C, and the supernatant was stored at −80 °C.
10
Ovalbumin-Induced Airway Sensitization
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Mice at 6 weeks of age were sensitized by intraperitoneal injections of 500 µl of phosphate-buffered saline (PBS) containing 10 µg of ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO) complexed with 1 mg aluminum hydroxide (Sigma-Aldrich) at days 0, 7 and 14. Sham immunization was performed by intraperitoneal injections of PBS alone. At days 21, 22 and 23, these sensitized mice were challenged for 30 min with PBS aerosol with or without 1% (w/v) OVA using an ultrasonic nebulizer (NE-U17; Omron, Kyoto, Japan).
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