Cells were chilled with ice for 15 minutes to detach from the culture tube and then placed into an Attofluor cell chamber (Molecular Probes) and incubated in a GasPak EZ anaerobic pouch (BD) or a Tri-gas incubator (Panasonic) set to 2.5% O2, 5% CO2 for 90 minutes at 37°C. For live imaging of Halo-tagged Delta-Giardin JaneliaFluor 646 HaloTag ligand (Promega, GA1120) or JaneliaFluor 549 HaloTag ligand (Promega, GA1110) were added to Attofluor cell chambers in growth media at a ratio of 1:1000 during the initial re-attachment step. Cells were then washed four times with HEPES-buffered saline (HBS: 137mM NaCl, 5mM KCl, 0.91mM Na2HpO4-heptahydrate, 5.55mM Glucose, 20mM HEPES, pH7). Live cell imaging was performed on a DeltaVision Elite microscope (GE) equipped with DIC optics, using a 100 × 1.4 NA or 60 × 1.42 NA objective, and a sCMOS 5.4 PCle air-cooled camera (PCO-TECH) and an Oko lab Bold Line stage-top, incubator, set to 37°C, 2.5% O2, and 5% CO2.
Gaspak ez anaerobic pouch
Manufactured by BD
Sourced in United States
The GasPak EZ anaerobic pouch is a self-contained system designed to create an anaerobic environment for the growth of anaerobic bacteria. The pouch incorporates a gas-generating system and an oxygen-absorbing system to establish and maintain the anaerobic conditions.
Automatically generated - may contain errors
Lab products found in correlation
Attofluor cell chamber, by Thermo Fisher Scientific (5 mentions)
Deltavision elite microscope, by GE Healthcare (5 mentions)
Tri gas incubator, by Panasonic (5 mentions)
Ultra low gelling agarose, by Merck Group (3 mentions)
Janelia fluor 549 halotag ligand, by Promega (1 mentions)
Janeliafluor 646 halotag ligand, by Promega (1 mentions)
Urea, by Hardy Diagnostics (1 mentions)
Sirtinol, by Merck Group (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Hk 2 cell, by American Type Culture Collection (1 mentions)
7 protocols using gaspak ez anaerobic pouch
1
Live Imaging of Halo-Tagged Delta-Giardin
2
Imaging Live Cells under Hypoxia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were chilled with ice for 15 min to detach them from the culture tube and then placed into an Attofluor cell chamber (Molecular Probes) and incubated in a GasPak EZ anaerobic pouch (BD) or a Tri-gas incubator (Panasonic) set to 2.5% O2, 5% CO2 for 90 min at 37°C. Cells were then washed four times with HEPES-buffered saline (137 mM NaCl, 5 mM KCl, 0.91 mM Na2HPO4-heptahydrate, 5.55 mM glucose, 20 mM HEPES, pH 7), overlaid with a mixture of 0.7% ultralow gelling agarose (Sigma A2576) melted in HEPES-buffered saline, cooled for 10 min at room temperature to solidify, and then imaged. Live cell imaging was performed on a DeltaVision Elite microscope (GE) equipped with differential interference contrast (DIC) optics, using a 100× 1.4 numerical aperture (NA) or 60× 1.42 NA lens objective and a scientific complementary metal oxide semiconductor (sCMOS) 5.4 PCle air-cooled camera (PCO-TECH).
3
Imaging Live Cells under Hypoxia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were chilled with ice for 15 min to detach them from the culture tube and then placed into an Attofluor cell chamber (Molecular Probes) and incubated in a GasPak EZ anaerobic pouch (BD) or a Tri-gas incubator (Panasonic) set to 2.5% O2, 5% CO2 for 90 min at 37°C. Cells were then washed four times with HEPES-buffered saline (137 mM NaCl, 5 mM KCl, 0.91 mM Na2HPO4-heptahydrate, 5.55 mM glucose, 20 mM HEPES, pH 7), overlaid with a mixture of 0.7% ultralow gelling agarose (Sigma A2576) melted in HEPES-buffered saline, cooled for 10 min at room temperature to solidify, and then imaged. Live cell imaging was performed on a DeltaVision Elite microscope (GE) equipped with differential interference contrast (DIC) optics, using a 100× 1.4 numerical aperture (NA) or 60× 1.42 NA lens objective and a scientific complementary metal oxide semiconductor (sCMOS) 5.4 PCle air-cooled camera (PCO-TECH).
4
Live Cell Imaging of Chilled Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were chilled with ice for 15 minutes to detach from the culture tube and then placed into an Attofluor cell chamber (Molecular Probes) and incubated in a GasPak EZ anaerobic pouch (BD) or a Tri-gas incubator (Panasonic) set to 2.5% O2, 5% CO2 for 90 minutes at 37°C. Cells were then washed four times with HEPES-buffered saline (HBS: 137mM NaCl, 5mM KCl, 0.91mM Na2HpO4-heptahydrate, 5.55mM Glucose, 20mM HEPES, pH7). Live cell imaging was performed on a DeltaVision Elite microscope (GE) equipped with DIC optics, using a 100 × 1.4 NA or 60 × 1.42 NA objective, and a sCMOS 5.4 PCle air-cooled camera (PCO-TECH).
5
Live-cell Imaging of Anaerobic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were chilled with ice for 15 minutes to detach from the culture tube and then placed into an Attofluor cell chamber (Molecular Probes) and incubated in a GasPak EZ anaerobic pouch (BD) or a Tri-gas incubator (Panasonic) set to 2.5% O2, 5% Co2 for 90 minutes at 37° C. Cells were then washed four times with HEPES-buffered saline (137mM NaCl, 5mM KCl, 0.91mM Na2HpO4-heptahydrate, 5. 55mM Glucose, 20mM HEPES, and pH7), overlaid with a mixture of 0.7% ultra-low gelling agarose (Sigma A2576) melted in HEPES-buffered saline, cooled for 10 minutes at room temperature to solidify, then imaged. Live cell imaging was performed on a DeltaVision Elite microscope (GE) equipped with DIC optics, using a 100X 1.4 NA or 60X 1.42 NA objective, and a sCMOS 5.4 PCle air-cooled camera (PCO-TECH).
6
Fetal Microbiome Sampling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Within 3 h of fetal delivery, ESwab samples for bacterial culture were processed in a biological safety cabinet by study personnel wearing a sterile surgical gown, mask, full hood, and powder-free exam gloves. Specifically, ESwab buffer solutions were added to SP4 broth with urea (Hardy Diagnostics, Santa Maria, CA) and were plated on blood agar (Trypticase soy agar with 5% sheep blood) and chocolate agar. Samples of the chorionic plate (amnion-chorion interface and the subchorion), villous tree, and fetal distal colon were inoculated on each culture medium. ESwab samples of the fetal proximal colon were inoculated on blood and chocolate agar but not SP4 broth. Blood and chocolate agar plates were incubated under aerobic (5% CO2) and anaerobic (BD GasPak EZ anaerobic pouch) atmospheres at 37°C for 7 days. SP4 broth was only incubated under aerobic conditions. Negative and positive (blood and chocolate agar inoculated with a human buccal ESwab) culture medium controls were incubated alongside the rhesus macaque samples for 7 days.
7
Hypoxia Response and Sirtuin Modulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HK2 cells purchased from American Type Culture Collection (Manassas, VA, USA) were cultured at 37°C in a 5% carbon dioxide (CO2) atmosphere in DMEM (Life Technologies, Carlsbad, NY, USA) mixed 1:1 (vol:vol) with F12 medium (Life Technologies) supplemented with 10% FBS (Life Technologies). Near confluent cells were incubated with serum‐free media for 24 hr to arrest and synchronize cell cycle. For hypoxic stimuli, cells were incubated in a hypoxic chamber using BD GAS‐PAK EZ anaerobic pouch (BD, Sparks, MD, USA) with 5% CO2/1% O2 and 94% N2 (v/v). For some experiments, cells were pretreated or treated with resveratrol (10 µM; Sigma‐Aldrich, St Louis, MO, USA) or sirtinol (10 µM; Sigma‐Aldrich) for the specified duration.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!