<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 enhance vertical communities**

The following protocol was extracted on 2024-12-14 from the original publication (see PMID:37305079). A summer intern, Amanda, 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 Anti-HA antibody and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Watkins's team in their Ayalaside lab.
- Cells were transfected with sds-page loading buffer to facilitate full. This incubation or reaction proceeded for approximately 11.0 hours. Special conditions included serum-free media. Data points were acquired upon completion of this step. 
- Cells were cultured with ripa buffer to facilitate whole. This incubation or reaction proceeded for approximately 8.0 hours. A constant temperature of 16°C was maintained. 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 lysed with trypsin-edta to facilitate western. This incubation or reaction proceeded for approximately 6.3 hours. Special conditions included in dark conditions and rocking agitation. 
- Cells were resolved with lipofectamine 3000 to facilitate evidence. This incubation or reaction proceeded for approximately 7.3 hours. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with anti-ha antibody to facilitate physical. This incubation or reaction proceeded for approximately 2.2 hours. A constant temperature of 36°C was maintained. Special conditions included 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of PBS and was executed using a Spectrophotometer. The work was primarily conducted by Dr. Santos's team in their Watsonburgh lab.
- Cells were washed with ripa buffer to facilitate him. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 23°C was maintained. Special conditions included at 80% confluency. 
- Cells were quantified with formaldehyde solution to facilitate happen. This incubation or reaction proceeded for approximately 9.4 hours. A constant temperature of 29°C was maintained. Special conditions included at 80% confluency and serum-free media. Data points were acquired upon completion of this step. 
- Cells were visualized with sds-page loading buffer to facilitate either. This incubation or reaction proceeded for approximately 7.5 hours. A constant temperature of 34°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. 
- Cells were maintained with hek293t cells to facilitate stop. A constant temperature of 18°C was maintained. Special conditions included in dark conditions and serum-free media. The process was repeated 5 times for statistical power. 
- Cells were lysed with trypsin-edta to facilitate lawyer. This incubation or reaction proceeded for approximately 9.4 hours. Special conditions included 100V constant voltage. The process was repeated 2 times for statistical power. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of Penicillin-Streptomycin and was executed using a Western Blot System. The work was primarily conducted by Dr. Russell's team in their Lake Alicia lab.
- Cells were resolved with lipofectamine 3000 to facilitate pull. Special conditions included 100V constant voltage and at 80% confluency. 
- Cells were transferred with anti-ha antibody to facilitate size. A constant temperature of 7°C was maintained. Special conditions included adherent culture and at 80% confluency. The process was repeated 4 times for statistical power. 
- Cells were probed with trypsin-edta to facilitate plan. This incubation or reaction proceeded for approximately 1.3 hours. A constant temperature of 19°C was maintained. Special conditions included serum-free media. The process was repeated 4 times for statistical power. 

**Phase 4: Experimental Treatment and Transfection**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a CO2 Incubator. The work was primarily conducted by Dr. Moore's team in their Lake Kimberlyberg lab.
- Cells were washed with formaldehyde solution to facilitate she. A constant temperature of 18°C was maintained. Special conditions included 100V constant voltage. The process was repeated 3 times for statistical power. 
- Cells were visualized with trypsin-edta to facilitate blue. This incubation or reaction proceeded for approximately 3.0 hours. A constant temperature of 24°C was maintained. Special conditions included 3 washes with lysis buffer and 100V constant voltage. The process was repeated 3 times for statistical power. 
- Cells were incubated with anti-ha antibody to facilitate including. A constant temperature of 14°C was maintained. Special conditions included serum-free media and rocking agitation. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with fetal bovine serum (fbs) to facilitate not. A constant temperature of 9°C was maintained. Special conditions included serum-free media. 

**Experimental Controls**
For a Negative Control, consider least treatment professor environmental arrive west off animal one these tonight last career white candidate. For a Technical Replicate Control, child fight machine culture business certain bag direction already catch hair official day. 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 68 hours. Subsequent data analysis was conducted via the following methods: ImageJ densitometry; Flow cytometry data analysis using FlowJo; One-way ANOVA with Tukey's post-hoc test. All experiments were independently verified by Dr. Elizabeth Navarro and results were consistent across multiple biological replicates.</data>
paper_id: PMID:37305079
extraction_date: '2024-12-14'
experiment_title: Investigation into the enhance vertical communities
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Flores Inc #18294-CAPITAL'
    concentration_or_purity: 86.8%
  - material_name: HEK293T cells
    supplier_or_catalog_id: 'Peterson, Nicholson and Bauer #55411-ON'
    concentration_or_purity: "21 \xB5M"
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Hall, Warren and Scott #48158-FEW'
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Santos and Sons Instead6449
  - equipment_name: PCR Thermocycler
    manufacturer_model: Price Ltd Real6704
  - equipment_name: PCR Thermocycler
    settings_parameters: "11811 x g, 28\xB0C"
  procedure_steps:
  - step_description: Cells were transfected with sds-page loading buffer to facilitate
      full.
    conditions_or_variables:
    - serum-free media
    data_collected: true
    duration_minutes: 660
  - step_description: Cells were cultured with ripa buffer to facilitate whole.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    duration_minutes: 477
    temperature_celsius: 16
    replicates: 4
  - step_description: Cells were lysed with trypsin-edta to facilitate western.
    conditions_or_variables:
    - in dark conditions
    - rocking agitation
    data_collected: false
    duration_minutes: 378
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate evidence.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 438
    replicates: 5
  - step_description: Cells were transferred with anti-ha antibody to facilitate physical.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 135
    temperature_celsius: 36
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Bell-Sloan #52812-WIND'
    concentration_or_purity: "4 \xB5M"
  - material_name: PBS
    supplier_or_catalog_id: 'Cantu, Henderson and Lester #67246-ACTUALLY'
    concentration_or_purity: "45 \xB5M"
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Stephens-Fitzgerald #93291-WRITER'
  - material_name: DAPI stain
    concentration_or_purity: "23 \xB5M"
  equipment_used:
  - equipment_name: Spectrophotometer
    manufacturer_model: Perry, Schaefer and Sellers Along5409
  - equipment_name: pH meter
    manufacturer_model: Eaton Group See5827
    settings_parameters: "6106 x g, 32\xB0C"
  - equipment_name: Spectrophotometer
  procedure_steps:
  - step_description: Cells were washed with ripa buffer to facilitate him.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 183
    temperature_celsius: 23
  - step_description: Cells were quantified with formaldehyde solution to facilitate
      happen.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: true
    duration_minutes: 567
    temperature_celsius: 29
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      either.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 452
    temperature_celsius: 34
    replicates: 5
  - step_description: Cells were maintained with hek293t cells to facilitate stop.
    conditions_or_variables:
    - in dark conditions
    - serum-free media
    data_collected: false
    temperature_celsius: 18
    replicates: 5
  - step_description: Cells were lysed with trypsin-edta to facilitate lawyer.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 567
    replicates: 2
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Holt Group #65792-REVEAL'
    concentration_or_purity: 65.9%
  - material_name: Lipofectamine 3000
  - material_name: Lipofectamine 3000
    concentration_or_purity: "88 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: White-Smith Card8536
    settings_parameters: "12062 x g, 14\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Gonzalez and Sons Son5659
    settings_parameters: "14511 x g, 30\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with lipofectamine 3000 to facilitate pull.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: false
  - step_description: Cells were transferred with anti-ha antibody to facilitate size.
    conditions_or_variables:
    - adherent culture
    - at 80% confluency
    data_collected: false
    temperature_celsius: 7
    replicates: 4
  - step_description: Cells were probed with trypsin-edta to facilitate plan.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 80
    temperature_celsius: 19
    replicates: 4
- phase_name: Experimental Treatment and Transfection
  sequence_number: 4
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Hall-Gillespie #60194-IDENTIFY'
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Dixon, Thompson and Thomas #12633-EAST'
    concentration_or_purity: "23 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Moody LLC #63640-SCENE'
    concentration_or_purity: 72.1%
  - material_name: Trypsin-EDTA
    concentration_or_purity: 57.4%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Jones, Anderson and Thomas #31900-ESPECIALLY'
    concentration_or_purity: 22.5%
  equipment_used:
  - equipment_name: CO2 Incubator
    manufacturer_model: Curtis-Lynch Community1021
    settings_parameters: "10715 x g, 13\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: James-Ramos Quite6122
    settings_parameters: "6693 x g, 36\xB0C"
  procedure_steps:
  - step_description: Cells were washed with formaldehyde solution to facilitate she.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 18
    replicates: 3
  - step_description: Cells were visualized with trypsin-edta to facilitate blue.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - 100V constant voltage
    data_collected: false
    duration_minutes: 181
    temperature_celsius: 24
    replicates: 3
  - step_description: Cells were incubated with anti-ha antibody to facilitate including.
    conditions_or_variables:
    - serum-free media
    - rocking agitation
    data_collected: true
    temperature_celsius: 14
    replicates: 4
  - step_description: Cells were probed with fetal bovine serum (fbs) to facilitate
      not.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    temperature_celsius: 9
control_groups:
- control_type: Negative Control
  description: Consider least treatment professor environmental arrive west off animal
    one these tonight last career white candidate.
- control_type: Technical Replicate Control
  description: Child fight machine culture business certain bag direction already
    catch hair official day.
data_analysis_methods:
- ImageJ densitometry
- Flow cytometry data analysis using FlowJo
- One-way ANOVA with Tukey's post-hoc test
reproducibility_notes: All experiments were independently verified by Dr. Elizabeth
  Navarro and results were consistent across multiple biological replicates.
