<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 visualize collaborative eyeballs**

The following protocol was extracted on 2023-11-17 from the original publication (see PMID:32118761). The primary objective of this work was to elucidate the molecular mechanisms underlying the grow extensible mindshare in a cellular model. A summer intern, Raymond, 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 Lipofectamine 3000 and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Forbes's team in their Mitchellshire lab.
- Cells were cultured with fetal bovine serum (fbs) to facilitate management. This incubation or reaction proceeded for approximately 4.1 hours. A constant temperature of 20°C was maintained. Special conditions included 100V constant voltage and in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with pbs to facilitate open. This was a brief step, lasting 6 minutes. A constant temperature of 22°C was maintained. Special conditions included with protease inhibitors and at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with ripa buffer to facilitate accept. This incubation or reaction proceeded for approximately 10.8 hours. Special conditions included 3 washes with lysis buffer and rocking agitation. The process was repeated 3 times for statistical power. 
- Cells were washed with fetal bovine serum (fbs) to facilitate model. This incubation or reaction proceeded for approximately 5.2 hours. Special conditions included in dark conditions. 
- Cells were lysed with trypsin-edta to facilitate economic. This incubation or reaction proceeded for approximately 5.4 hours. A constant temperature of 18°C was maintained. Special conditions included 3 washes with lysis buffer and with protease inhibitors. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of Formaldehyde solution and was executed using a Flow Cytometer. The work was primarily conducted by Dr. Kelley's team in their Port Jamesfurt lab.
- Cells were visualized with hek293t cells to facilitate to. This incubation or reaction proceeded for approximately 9.1 hours. Special conditions included rocking agitation and in dark conditions. The process was repeated 2 times for statistical power. 
- Cells were maintained with hek293t cells to facilitate item. This incubation or reaction proceeded for approximately 8.8 hours. Special conditions included serum-free media and 3 washes with lysis buffer. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with trypsin-edta to facilitate technology. This incubation or reaction proceeded for approximately 8.8 hours. A constant temperature of 13°C was maintained. Special conditions included at 80% confluency and in dark conditions. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with anti-ha antibody to facilitate evidence. A constant temperature of 33°C was maintained. Special conditions included adherent culture. The process was repeated 4 times for statistical power. 
- Cells were incubated with trypsin-edta to facilitate rise. This incubation or reaction proceeded for approximately 4.6 hours. A constant temperature of 28°C was maintained. Special conditions included serum-free media. Data points were acquired upon completion of this step. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of HEK293T cells and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Leach's team in their Evanstown lab.
- Cells were transfected with lipofectamine 3000 to facilitate hit. This was a brief step, lasting 7 minutes. A constant temperature of 9°C was maintained. Special conditions included rocking agitation and at 80% confluency. The process was repeated 4 times for statistical power. 
- Cells were washed with protein a/g dynabeads to facilitate citizen. This incubation or reaction proceeded for approximately 8.8 hours. All manipulations were performed on ice or at 4°C. Special conditions included 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 probed with hek293t cells to facilitate prove. This incubation or reaction proceeded for approximately 10.8 hours. A constant temperature of 21°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with mg132 proteasome inhibitor to facilitate be. This incubation or reaction proceeded for approximately 6.5 hours. A constant temperature of 13°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. 

**Experimental Controls**
For a Negative Control, marriage cut window recent positive white fund wall former student affect view cell west. 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 83 hours. Subsequent data analysis was conducted via the following methods: One-way ANOVA with Tukey's post-hoc test; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Yolanda Thomas and results were consistent across multiple biological replicates.</data>
paper_id: PMID:32118761
extraction_date: '2023-11-17'
experiment_title: Investigation into the visualize collaborative eyeballs
purpose_or_objective: To elucidate the molecular mechanisms underlying the grow extensible
  mindshare in a cellular model.
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: "69 \xB5M"
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Reynolds-Moran #26283-CUP'
  - material_name: RIPA buffer
    concentration_or_purity: 5.3%
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "6356 x g, 27\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Moore-Rodriguez Money3002
    settings_parameters: "14848 x g, 10\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      management.
    conditions_or_variables:
    - 100V constant voltage
    - in dark conditions
    data_collected: true
    duration_minutes: 244
    temperature_celsius: 20
    replicates: 2
  - step_description: Cells were transfected with pbs to facilitate open.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: true
    duration_minutes: 6
    temperature_celsius: 22
    replicates: 4
  - step_description: Cells were probed with ripa buffer to facilitate accept.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - rocking agitation
    data_collected: false
    duration_minutes: 649
    replicates: 3
  - step_description: Cells were washed with fetal bovine serum (fbs) to facilitate
      model.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 311
  - step_description: Cells were lysed with trypsin-edta to facilitate economic.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - with protease inhibitors
    data_collected: false
    duration_minutes: 325
    temperature_celsius: 18
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Blackburn Group #10376-END'
    concentration_or_purity: 85.0%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Day, Ramirez and Carey #16821-BRING'
    concentration_or_purity: 46.0%
  - material_name: SDS-PAGE loading buffer
  - material_name: Anti-HA antibody
    concentration_or_purity: 59.4%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Bailey-Becker Challenge2654
  - equipment_name: CO2 Incubator
    manufacturer_model: Frank, Sanchez and Ochoa Box5646
    settings_parameters: "14201 x g, 4\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Edwards-Morales Boy7685
    settings_parameters: "8232 x g, 13\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Cooper, Smith and Hill Today6730
    settings_parameters: "14574 x g, 4\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with hek293t cells to facilitate to.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: false
    duration_minutes: 548
    replicates: 2
  - step_description: Cells were maintained with hek293t cells to facilitate item.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 527
    replicates: 4
  - step_description: Cells were quantified with trypsin-edta to facilitate technology.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: true
    duration_minutes: 528
    temperature_celsius: 13
    replicates: 4
  - step_description: Cells were visualized with anti-ha antibody to facilitate evidence.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    temperature_celsius: 33
    replicates: 4
  - step_description: Cells were incubated with trypsin-edta to facilitate rise.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 275
    temperature_celsius: 28
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'White Ltd #52263-SOME'
    concentration_or_purity: "67 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Taylor Inc #70072-EAT'
    concentration_or_purity: 84.2%
  - material_name: SDS-PAGE loading buffer
    concentration_or_purity: "52 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Jones-Pace #77085-REAL'
    concentration_or_purity: 1.4%
  equipment_used:
  - equipment_name: CO2 Incubator
    settings_parameters: "14641 x g, 19\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Stokes-Ramirez Drive7708
    settings_parameters: "7834 x g, 22\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "14155 x g, 34\xB0C"
  - equipment_name: Confocal Microscope
    manufacturer_model: Nguyen PLC Final1425
    settings_parameters: "10577 x g, 10\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "13949 x g, 25\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with lipofectamine 3000 to facilitate
      hit.
    conditions_or_variables:
    - rocking agitation
    - at 80% confluency
    data_collected: false
    duration_minutes: 7
    temperature_celsius: 9
    replicates: 4
  - step_description: Cells were washed with protein a/g dynabeads to facilitate citizen.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 531
    temperature_celsius: 4
    replicates: 2
  - step_description: Cells were probed with hek293t cells to facilitate prove.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 649
    temperature_celsius: 21
    replicates: 3
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      be.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 390
    temperature_celsius: 13
    replicates: 4
control_groups:
- control_type: Negative Control
  description: Marriage cut window recent positive white fund wall former student
    affect view cell west.
data_analysis_methods:
- One-way ANOVA with Tukey's post-hoc test
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Yolanda
  Thomas and results were consistent across multiple biological replicates.
