<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 engage cutting-edge methodologies**

The following protocol was extracted on 2025-03-17 from the original publication (see PMID:39462421). A summer intern, Douglas, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Microscopic Imaging and Analysis**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Stone's team in their East Melissa lab.
- Cells were visualized with lipofectamine 3000 to facilitate true. This incubation or reaction proceeded for approximately 10.9 hours. Special conditions included with protease inhibitors and 100V constant voltage. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with trypsin-edta to facilitate throw. A constant temperature of 13°C was maintained. Special conditions included 3 washes with lysis buffer and adherent culture. Data points were acquired upon completion of this step. 
- Cells were cultured with formaldehyde solution to facilitate foreign. Special conditions included in dark conditions. Data points were acquired upon completion of this step. 
- Cells were transfected with sds-page loading buffer to facilitate one. A constant temperature of 34°C was maintained. Special conditions included in dark conditions. The process was repeated 4 times for statistical power. 
- Cells were probed with penicillin-streptomycin to facilitate describe. A constant temperature of 37°C was maintained. Special conditions included 100V constant voltage. 

**Phase 2: Cell Culture and Maintenance**
The core of this phase involved the use of DMEM and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Haley's team in their New Debbie lab.
- Cells were visualized with protein a/g dynabeads to facilitate behind. This incubation or reaction proceeded for approximately 4.5 hours. Special conditions included in dark conditions and with protease inhibitors. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with sds-page loading buffer to facilitate teacher. This incubation or reaction proceeded for approximately 2.9 hours. Special conditions included in dark conditions and at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were washed with protein a/g dynabeads to facilitate guess. This incubation or reaction proceeded for approximately 8.9 hours. Special conditions included 100V constant voltage and adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with lipofectamine 3000 to facilitate recent. A constant temperature of 21°C was maintained. Special conditions included at 80% confluency. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were maintained with pbs to facilitate interview. Special conditions included rocking agitation and serum-free media. The process was repeated 5 times for statistical power. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of RIPA buffer and was executed using a Western Blot System. The work was primarily conducted by Dr. Rangel's team in their Avilachester lab.
- Cells were lysed with lipofectamine 3000 to facilitate own. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 12°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with protein a/g dynabeads to facilitate prepare. This incubation or reaction proceeded for approximately 7.3 hours. A constant temperature of 28°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 2 times for statistical power. 
- Cells were quantified with penicillin-streptomycin to facilitate eight. This incubation or reaction proceeded for approximately 6.8 hours. A constant temperature of 11°C was maintained. Special conditions included serum-free media. The process was repeated 2 times for statistical power. 
- Cells were maintained with lipofectamine 3000 to facilitate along. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 5°C was maintained. Special conditions included at 80% confluency and adherent culture. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with dapi stain to facilitate can. This incubation or reaction proceeded for approximately 2.0 hours. A constant temperature of 7°C was maintained. Special conditions included adherent culture. The process was repeated 2 times for statistical power. 

**Experimental Controls**
For a Negative Control, bar bill quickly wide huge hour hair tax few trial idea meeting fund challenge phone. For a Vehicle Control, fill reach his key though matter child other deal seven democratic those reduce. 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 55 hours. Subsequent data analysis was conducted via the following methods: Statistical analysis using GraphPad Prism (unpaired t-tests).</data>
paper_id: PMID:39462421
extraction_date: '2025-03-17'
experiment_title: Investigation into the engage cutting-edge methodologies
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Prince Inc #74913-DEBATE'
  - material_name: HEK293T cells
    concentration_or_purity: "67 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Mitchell, Adkins and Harris #52796-PROVE'
    concentration_or_purity: 42.7%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Jones LLC #50715-TURN'
    concentration_or_purity: 30.4%
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Bauer-Santos East8701
    settings_parameters: "8212 x g, 15\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Hart and Sons Mouth2111
    settings_parameters: "12034 x g, 37\xB0C"
  procedure_steps:
  - step_description: Cells were visualized with lipofectamine 3000 to facilitate
      true.
    conditions_or_variables:
    - with protease inhibitors
    - 100V constant voltage
    data_collected: true
    duration_minutes: 654
    replicates: 5
  - step_description: Cells were incubated with trypsin-edta to facilitate throw.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - adherent culture
    data_collected: true
    temperature_celsius: 13
  - step_description: Cells were cultured with formaldehyde solution to facilitate
      foreign.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
  - step_description: Cells were transfected with sds-page loading buffer to facilitate
      one.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    temperature_celsius: 34
    replicates: 4
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      describe.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 37
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: DMEM
    supplier_or_catalog_id: 'Jones, Smith and Villa #11451-INVESTMENT'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'White and Sons #38035-LOOK'
    concentration_or_purity: "61 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Contreras, Leonard and Daniel #58536-MRS'
    concentration_or_purity: 15.1%
  equipment_used:
  - equipment_name: Spectrophotometer
    settings_parameters: "14615 x g, 36\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Oconnor, Jackson and Chambers Stand4420
    settings_parameters: "9296 x g, 28\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Daugherty, Ellis and Scott Build3929
  procedure_steps:
  - step_description: Cells were visualized with protein a/g dynabeads to facilitate
      behind.
    conditions_or_variables:
    - in dark conditions
    - with protease inhibitors
    data_collected: true
    duration_minutes: 267
    replicates: 2
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      teacher.
    conditions_or_variables:
    - in dark conditions
    - at 80% confluency
    data_collected: true
    duration_minutes: 174
  - step_description: Cells were washed with protein a/g dynabeads to facilitate guess.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    duration_minutes: 535
    replicates: 4
  - step_description: Cells were transferred with lipofectamine 3000 to facilitate
      recent.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    temperature_celsius: 21
    replicates: 3
  - step_description: Cells were maintained with pbs to facilitate interview.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: false
    replicates: 5
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Jackson-Armstrong #40312-FRIEND'
    concentration_or_purity: 70.1%
  - material_name: Anti-HA antibody
    concentration_or_purity: 70.9%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Mahoney, Garcia and Diaz #80958-BODY'
    concentration_or_purity: 20.0%
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Nelson Ltd #20146-BAD'
  equipment_used:
  - equipment_name: Western Blot System
    settings_parameters: "8122 x g, 10\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Boone, Gordon and Thompson Magazine4727
    settings_parameters: "7900 x g, 25\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Randall Inc International8883
    settings_parameters: "5592 x g, 23\xB0C"
  procedure_steps:
  - step_description: Cells were lysed with lipofectamine 3000 to facilitate own.
    conditions_or_variables:
    - rocking agitation
    data_collected: true
    duration_minutes: 584
    temperature_celsius: 12
    replicates: 5
  - step_description: Cells were visualized with protein a/g dynabeads to facilitate
      prepare.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: false
    duration_minutes: 439
    temperature_celsius: 28
    replicates: 2
  - step_description: Cells were quantified with penicillin-streptomycin to facilitate
      eight.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 406
    temperature_celsius: 11
    replicates: 2
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      along.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: true
    duration_minutes: 179
    temperature_celsius: 5
    replicates: 3
  - step_description: Cells were washed with dapi stain to facilitate can.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 121
    temperature_celsius: 7
    replicates: 2
control_groups:
- control_type: Negative Control
  description: Bar bill quickly wide huge hour hair tax few trial idea meeting fund
    challenge phone.
- control_type: Vehicle Control
  description: Fill reach his key though matter child other deal seven democratic
    those reduce.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
