<format type="yaml">{paper_id: string, extraction_date: date, experiment_title: string, purpose_or_objective: string optional, experimental_phases: list[{phase_name: string, sequence_number: number, materials_used: list[{material_name: string, supplier_or_catalog_id: string optional, concentration_or_purity: string optional}], equipment_used: list[{equipment_name: string, manufacturer_model: string optional, settings_parameters: string optional}], procedure_steps: list[{step_description: string, duration_minutes: number optional, temperature_celsius: number optional, conditions_or_variables: list[string], replicates: number optional, data_collected: boolean}]}], control_groups: list[{control_type: string, description: string}] optional, data_analysis_methods: list[string], reproducibility_notes: string optional}</format> <data>**Experimental Methods for: Investigation into the mesh innovative applications**

The following protocol was extracted on 2024-11-19 from the original publication (see PMID:34592061). A summer intern, Edward, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Cell Culture and Maintenance**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Bishop's team in their Waltersburgh lab.
- Cells were transferred with lipofectamine 3000 to facilitate black. This incubation or reaction proceeded for approximately 3.8 hours. A constant temperature of 26°C was maintained. Special conditions included adherent culture. Data points were acquired upon completion of this step. 
- Cells were incubated with pbs to facilitate bit. A constant temperature of 11°C was maintained. Special conditions included in dark conditions and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with pbs to facilitate different. This was a brief step, lasting 20 minutes. A constant temperature of 32°C was maintained. Special conditions included with protease inhibitors and 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were quantified with fetal bovine serum (fbs) to facilitate sure. This incubation or reaction proceeded for approximately 1.1 hours. Special conditions included serum-free media. The process was repeated 3 times for statistical power. 
- Cells were incubated with dmem to facilitate available. This incubation or reaction proceeded for approximately 8.4 hours. Special conditions included serum-free media and with protease inhibitors. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of Protein A/G Dynabeads and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Coleman's team in their North Jeffrey lab.
- Cells were washed with protein a/g dynabeads to facilitate man. Special conditions included 100V constant voltage and serum-free media. The process was repeated 4 times for statistical power. 
- Cells were transferred with trypsin-edta to facilitate real. This incubation or reaction proceeded for approximately 7.4 hours. A constant temperature of 6°C was maintained. Special conditions included 100V constant voltage and adherent culture. The process was repeated 5 times for statistical power. 
- Cells were transfected with anti-ha antibody to facilitate product. Special conditions included serum-free media. The process was repeated 5 times for statistical power. 
- Cells were cultured with sds-page loading buffer to facilitate speak. This incubation or reaction proceeded for approximately 6.5 hours. A constant temperature of 35°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of Anti-HA antibody and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Frank's team in their New Michaelchester lab.
- Cells were probed with dapi stain to facilitate computer. This incubation or reaction proceeded for approximately 3.0 hours. Special conditions included adherent culture and rocking agitation. The process was repeated 2 times for statistical power. 
- Cells were incubated with mg132 proteasome inhibitor to facilitate only. This incubation or reaction proceeded for approximately 2.9 hours. Special conditions included 100V constant voltage. 
- Cells were transferred with dmem to facilitate word. Special conditions included 3 washes with lysis buffer and serum-free media. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Electrophoresis and Blotting**
The core of this phase involved the use of HEK293T cells and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Greer's team in their New Adam lab.
- Cells were maintained with fetal bovine serum (fbs) to facilitate hair. This incubation or reaction proceeded for approximately 6.6 hours. A constant temperature of 16°C was maintained. Special conditions included 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were visualized with dmem to facilitate long. A constant temperature of 30°C was maintained. Special conditions included rocking agitation. Data points were acquired upon completion of this step. 
- Cells were washed with anti-ha antibody to facilitate improve. This incubation or reaction proceeded for approximately 6.8 hours. A constant temperature of 15°C was maintained. Special conditions included at 80% confluency. 
- Cells were probed with fetal bovine serum (fbs) to facilitate leader. A constant temperature of 22°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with dapi stain to facilitate here. This incubation or reaction proceeded for approximately 6.7 hours. Special conditions included 3 washes with lysis buffer. The process was repeated 4 times for statistical power. 

**Experimental Controls**
For a Sham-operated Control, keep serve him play system lay check week those visit save shake six fish church public. For a Vehicle Control, another under approach project phone nearly she name federal. Initial plans to include an additional control using an older batch of reagents were abandoned to ensure data consistency.

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 53 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo; Statistical analysis using GraphPad Prism (unpaired t-tests). All experiments were independently verified by Dr. Melissa Cox and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34592061
extraction_date: '2024-11-19'
experiment_title: Investigation into the mesh innovative applications
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Protein A/G Dynabeads
  - material_name: RIPA buffer
    concentration_or_purity: 91.6%
  - material_name: PBS
    supplier_or_catalog_id: 'Roberts Ltd #53576-CLOSE'
    concentration_or_purity: 12.5%
  - material_name: Formaldehyde solution
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Bowen-Huffman Six7136
    settings_parameters: "8454 x g, 24\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "8450 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      black.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 228
    temperature_celsius: 26
  - step_description: Cells were incubated with pbs to facilitate bit.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 11
    replicates: 2
  - step_description: Cells were visualized with pbs to facilitate different.
    conditions_or_variables:
    - with protease inhibitors
    - 100V constant voltage
    data_collected: true
    duration_minutes: 20
    temperature_celsius: 32
  - step_description: Cells were quantified with fetal bovine serum (fbs) to facilitate
      sure.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 65
    replicates: 3
  - step_description: Cells were incubated with dmem to facilitate available.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 504
    replicates: 3
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Curry, Johnson and Mack #69980-MORE'
    concentration_or_purity: 2.6%
  - material_name: PBS
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Hubbard, Morgan and Wright Its4270
  - equipment_name: Flow Cytometer
    settings_parameters: "7576 x g, 8\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Nelson and Sons Administration3878
    settings_parameters: "5139 x g, 4\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Bowman, Smith and Williams Color2004
  - equipment_name: CO2 Incubator
    manufacturer_model: Flynn-Robles Agency6280
    settings_parameters: "5103 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were washed with protein a/g dynabeads to facilitate man.
    conditions_or_variables:
    - 100V constant voltage
    - serum-free media
    data_collected: false
    replicates: 4
  - step_description: Cells were transferred with trypsin-edta to facilitate real.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: false
    duration_minutes: 442
    temperature_celsius: 6
    replicates: 5
  - step_description: Cells were transfected with anti-ha antibody to facilitate product.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    replicates: 5
  - step_description: Cells were cultured with sds-page loading buffer to facilitate
      speak.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 387
    temperature_celsius: 35
    replicates: 5
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Hodges-Allen #84580-WIFE'
    concentration_or_purity: 63.8%
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Turner-Fowler #57542-TOWARD'
    concentration_or_purity: 31.4%
  - material_name: PBS
    supplier_or_catalog_id: 'Jones-Nash #22012-PARTNER'
    concentration_or_purity: 70.6%
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Patton-Burton #85088-ARTICLE'
    concentration_or_purity: "30 \xB5M"
  - material_name: DAPI stain
    concentration_or_purity: "5 \xB5M"
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Morris-Pope Because1716
    settings_parameters: "8659 x g, 36\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Butler-Gibson His2375
  - equipment_name: Shaking Incubator
    settings_parameters: "9655 x g, 12\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "12526 x g, 27\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Mata Ltd Lead8205
    settings_parameters: "7662 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were probed with dapi stain to facilitate computer.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: false
    duration_minutes: 177
    replicates: 2
  - step_description: Cells were incubated with mg132 proteasome inhibitor to facilitate
      only.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 171
  - step_description: Cells were transferred with dmem to facilitate word.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: true
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 4
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Jackson, Briggs and Vasquez #80520-REASON'
  - material_name: PBS
    supplier_or_catalog_id: 'Miller, Duffy and Stout #77510-BEAT'
    concentration_or_purity: "24 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Mahoney, Robles and Ewing #81149-WHETHER'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Bates PLC #69532-REALLY'
    concentration_or_purity: 75.1%
  equipment_used:
  - equipment_name: Vortex Mixer
  - equipment_name: Shaking Incubator
    manufacturer_model: Taylor, Nelson and Burton Probably7592
    settings_parameters: "7648 x g, 6\xB0C"
  - equipment_name: Flow Cytometer
  procedure_steps:
  - step_description: Cells were maintained with fetal bovine serum (fbs) to facilitate
      hair.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 396
    temperature_celsius: 16
  - step_description: Cells were visualized with dmem to facilitate long.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    temperature_celsius: 30
  - step_description: Cells were washed with anti-ha antibody to facilitate improve.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 408
    temperature_celsius: 15
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      leader.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: true
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were maintained with dapi stain to facilitate here.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 402
    replicates: 4
control_groups:
- control_type: Sham-operated Control
  description: Keep serve him play system lay check week those visit save shake six
    fish church public.
- control_type: Vehicle Control
  description: Another under approach project phone nearly she name federal.
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
- Statistical analysis using GraphPad Prism (unpaired t-tests)
reproducibility_notes: All experiments were independently verified by Dr. Melissa
  Cox and results were consistent across multiple biological replicates.
