Prior to euthanasia, blood was collected in non-heparinized capillary tubes (Fisher Scientific) and serum was collected as previously described (48 (link)). Lungs were homogenized using a Dounce tissue homogenizer and homogenates were purified by centrifugation. Serum and lung lysates were stored at −80°C until analysis. Serum and lung lysate samples were analyzed using the Bio-Plex Pro™ Mouse Chemokine Panel 33-Plex (Bio-Rad, Hercules, CA) per the manufacturer’s instructions. Data were acquired on a Bio-Plex 200 (Bio-Rad).
Non heparinized capillary tubes
Manufactured by Thermo Fisher Scientific
Non-heparinized capillary tubes are laboratory equipment used for the collection and storage of small volumes of liquid samples. They are made of glass or plastic and have a narrow, cylindrical shape. These tubes do not contain the anticoagulant heparin, which is typically used to prevent blood clotting.
Automatically generated - may contain errors
Lab products found in correlation
Heparinized capillary tube, by Thermo Fisher Scientific (2 mentions)
Bio plex pro mouse chemokine panel 33 plex, by Bio-Rad (1 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Dnase 1, by MP Biomedicals (1 mentions)
Collagenase type 3, by MP Biomedicals (1 mentions)
Serum collection tube, by BD (1 mentions)
Edta coated tube, by BD (1 mentions)
3 protocols using non heparinized capillary tubes
1
Serum and Lung Cytokine Profiling
2
Blood, BAL, and Tissue Processing Protocol
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Prior to euthanasia, blood was collected in heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA) for subsequent single-cell analysis by flow cytometry and non-heparinized capillary tubes (Fisher Scientific) for serum collection. For cell harvests, these blood samples were then treated with ammonium-chloride-potassium lysis buffer for 5 min at room temperature and washed 1X with flow cytometry staining buffer. For serum collection, blood samples were left at room temperature for 30 min, centrifuged at 16,000 × g for 20 min, and then collected and stored at −20°C until analysis.
Bronchial alveolar lavage (BAL) fluid was collected using a protocol modified from (35 (link)). Briefly, the tracheae were cannulated with a 22-gage catheter tube (attached to a 5cc syringe) and then washed once with 1 mL of sterile PBS. Samples were stored at −20°C until analysis.
For preparation of cells from lungs and spleens, these organs were harvested after the collection of BAL fluid, digested for 30 min at 37°C in media containing 1 mg/mL Collagenase (Type 3; MP Biomedicals, Solon, OH) and 0.02 mg/mL DNase-I (MP Biomedicals), and then pressed through wire mesh to obtain a single cell suspension.
Bronchial alveolar lavage (BAL) fluid was collected using a protocol modified from (35 (link)). Briefly, the tracheae were cannulated with a 22-gage catheter tube (attached to a 5cc syringe) and then washed once with 1 mL of sterile PBS. Samples were stored at −20°C until analysis.
For preparation of cells from lungs and spleens, these organs were harvested after the collection of BAL fluid, digested for 30 min at 37°C in media containing 1 mg/mL Collagenase (Type 3; MP Biomedicals, Solon, OH) and 0.02 mg/mL DNase-I (MP Biomedicals), and then pressed through wire mesh to obtain a single cell suspension.
3
Bile and Blood Collection in Mice
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For mice from which bile was not collected (Fig. 1 , S1 , 5a –d ), mice were anesthetized by intraperitoneal injection of ketamine and xylazine. Blood was then collected by retro-orbital puncture into EDTA-coated tubes (BD) using heparinized capillary tubes (Fisher) then into serum collection tubes (BD) using nonheparinized capillary tubes (Fisher). Mice were euthanized by cervical dislocation and tissues collected for metal, DNA, RNA, and protein analysis. (Gastrointestinal tracts were washed of lumenal contents.)
For all other mice, mice were anesthetized with isoflurane then underwent bile, blood, and tissue collection. Bile was collected surgically by ligation of the common bile duct, cannulation of the gallbladder, and collection over 60 minutes as previously described(6 (link)). Bile volumes were measured every five minutes to permit calculation of bile flow rates. Blood was then collected by cardiac puncture, and mice were transcardially perfused with PBS to remove blood from tissues. Tissues were then collected and processed as above.
For all other mice, mice were anesthetized with isoflurane then underwent bile, blood, and tissue collection. Bile was collected surgically by ligation of the common bile duct, cannulation of the gallbladder, and collection over 60 minutes as previously described(6 (link)). Bile volumes were measured every five minutes to permit calculation of bile flow rates. Blood was then collected by cardiac puncture, and mice were transcardially perfused with PBS to remove blood from tissues. Tissues were then collected and processed as above.
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