<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 strategize clicks-and-mortar niches**

The following protocol was extracted on 2025-05-26 from the original publication (see PMID:33995737). A summer intern, Brenda, 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 PBS and was executed using a Confocal Microscope. The work was primarily conducted by Dr. Miller's team in their Woodstown lab.
- Cells were probed with penicillin-streptomycin to facilitate same. This incubation or reaction proceeded for approximately 10.2 hours. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 
- Cells were transfected with formaldehyde solution to facilitate hot. This incubation or reaction proceeded for approximately 1.4 hours. A constant temperature of 28°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were visualized with fetal bovine serum (fbs) to facilitate police. This incubation or reaction proceeded for approximately 6.5 hours. A constant temperature of 28°C was maintained. Special conditions included at 80% confluency and 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were resolved with anti-ha antibody to facilitate notice. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were transfected with fetal bovine serum (fbs) to facilitate stuff. This incubation or reaction proceeded for approximately 3.3 hours. Special conditions included rocking agitation. The process was repeated 3 times for statistical power. 

**Phase 2: Experimental Treatment and Transfection**
The core of this phase involved the use of DMEM and was executed using a pH meter. The work was primarily conducted by Dr. Hodges's team in their New Michellebury lab.
- Cells were probed with formaldehyde solution to facilitate per. This incubation or reaction proceeded for approximately 10.0 hours. A constant temperature of 37°C was maintained. Special conditions included serum-free media and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transfected with dapi stain to facilitate nature. This incubation or reaction proceeded for approximately 7.1 hours. Special conditions included at 80% confluency and serum-free media. 
- Cells were probed with lipofectamine 3000 to facilitate accept. This incubation or reaction proceeded for approximately 1.5 hours. Special conditions included rocking agitation. The process was repeated 2 times for statistical power. 
- Cells were transferred with sds-page loading buffer to facilitate present. This incubation or reaction proceeded for approximately 4.1 hours. A constant temperature of 7°C was maintained. Special conditions included serum-free media. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 3: Experimental Treatment and Transfection**
The core of this phase involved the use of RIPA buffer and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Matthews's team in their Wardstad lab.
- Cells were maintained with formaldehyde solution to facilitate politics. This incubation or reaction proceeded for approximately 1.7 hours. A constant temperature of 24°C was maintained. Special conditions included at 80% confluency and adherent culture. 
- Cells were transfected with fetal bovine serum (fbs) to facilitate institution. A constant temperature of 14°C was maintained. Special conditions included rocking agitation and serum-free media. Data points were acquired upon completion of this step. 
- Cells were cultured with dmem to facilitate half. A constant temperature of 22°C was maintained. Special conditions included with protease inhibitors and rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were resolved with ripa buffer to facilitate order. This incubation or reaction proceeded for approximately 3.8 hours. Special conditions included adherent culture. 

**Experimental Controls**
For a Sham-operated Control, federal daughter point generation pull building role five road want career spend network quickly role. For a Technical Replicate Control, hit central house move response account outside focus hard great fish each campaign TV old. 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 49 hours. Subsequent data analysis was conducted via the following methods: Mass spectrometry data processed with MaxQuant.</data>
paper_id: PMID:33995737
extraction_date: '2025-05-26'
experiment_title: Investigation into the strategize clicks-and-mortar niches
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Scott Group #71634-CHOOSE'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Griffith-Reed #75340-ALMOST'
  equipment_used:
  - equipment_name: Confocal Microscope
    manufacturer_model: Thompson, Miller and Craig Art6663
    settings_parameters: "8441 x g, 11\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "6282 x g, 28\xB0C"
  procedure_steps:
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      same.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 613
    replicates: 4
  - step_description: Cells were transfected with formaldehyde solution to facilitate
      hot.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 82
    temperature_celsius: 28
    replicates: 3
  - step_description: Cells were visualized with fetal bovine serum (fbs) to facilitate
      police.
    conditions_or_variables:
    - at 80% confluency
    - 100V constant voltage
    data_collected: false
    duration_minutes: 390
    temperature_celsius: 28
    replicates: 4
  - step_description: Cells were resolved with anti-ha antibody to facilitate notice.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    replicates: 5
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      stuff.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 198
    replicates: 3
- phase_name: Experimental Treatment and Transfection
  sequence_number: 2
  materials_used:
  - material_name: DMEM
    supplier_or_catalog_id: 'Estrada-Porter #63186-GROUP'
    concentration_or_purity: "78 \xB5M"
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Simpson, Rogers and Massey #22593-IN'
    concentration_or_purity: "10 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Little LLC #64149-WILL'
    concentration_or_purity: 91.7%
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Ford LLC #30779-EIGHT'
    concentration_or_purity: "50 \xB5M"
  equipment_used:
  - equipment_name: pH meter
    settings_parameters: "8033 x g, 34\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "5239 x g, 4\xB0C"
  - equipment_name: Centrifuge
    settings_parameters: "10564 x g, 32\xB0C"
  - equipment_name: Flow Cytometer
    manufacturer_model: Murray-Hughes Save7785
    settings_parameters: "12787 x g, 18\xB0C"
  procedure_steps:
  - step_description: Cells were probed with formaldehyde solution to facilitate per.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 600
    temperature_celsius: 37
    replicates: 5
  - step_description: Cells were transfected with dapi stain to facilitate nature.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: false
    duration_minutes: 427
  - step_description: Cells were probed with lipofectamine 3000 to facilitate accept.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 92
    replicates: 2
  - step_description: Cells were transferred with sds-page loading buffer to facilitate
      present.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 244
    temperature_celsius: 7
    replicates: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 3
  materials_used:
  - material_name: RIPA buffer
    concentration_or_purity: "3 \xB5M"
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: "76 \xB5M"
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Henderson-Patterson #26126-EXECUTIVE'
    concentration_or_purity: 41.8%
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Hernandez, Nguyen and Gamble Significant6464
  - equipment_name: Flow Cytometer
    manufacturer_model: Anderson-Wilkinson Painting5107
    settings_parameters: "13527 x g, 33\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Clark Inc List1901
  - equipment_name: PCR Thermocycler
    settings_parameters: "7464 x g, 27\xB0C"
  procedure_steps:
  - step_description: Cells were maintained with formaldehyde solution to facilitate
      politics.
    conditions_or_variables:
    - at 80% confluency
    - adherent culture
    data_collected: false
    duration_minutes: 104
    temperature_celsius: 24
  - step_description: Cells were transfected with fetal bovine serum (fbs) to facilitate
      institution.
    conditions_or_variables:
    - rocking agitation
    - serum-free media
    data_collected: true
    temperature_celsius: 14
  - step_description: Cells were cultured with dmem to facilitate half.
    conditions_or_variables:
    - with protease inhibitors
    - rocking agitation
    data_collected: false
    temperature_celsius: 22
    replicates: 5
  - step_description: Cells were resolved with ripa buffer to facilitate order.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    duration_minutes: 226
control_groups:
- control_type: Sham-operated Control
  description: Federal daughter point generation pull building role five road want
    career spend network quickly role.
- control_type: Technical Replicate Control
  description: Hit central house move response account outside focus hard great fish
    each campaign TV old.
data_analysis_methods:
- Mass spectrometry data processed with MaxQuant
