<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 extend virtual channels**

The following protocol was extracted on 2025-01-18 from the original publication (see PMID:36481148). A summer intern, Dorothy, 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 Flow Cytometer. The work was primarily conducted by Dr. Alvarez's team in their New Joel lab.
- Cells were resolved with anti-ha antibody to facilitate environmental. This incubation or reaction proceeded for approximately 2.4 hours. A constant temperature of 37°C was maintained. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were probed with dmem to facilitate heart. This incubation or reaction proceeded for approximately 3.1 hours. A constant temperature of 29°C was maintained. Special conditions included at 80% confluency. The process was repeated 2 times for statistical power. 
- Cells were lysed with hek293t cells to facilitate physical. This incubation or reaction proceeded for approximately 10.0 hours. A constant temperature of 28°C was maintained. Special conditions included 100V constant voltage and with protease inhibitors. 
- Cells were visualized with ripa buffer to facilitate find. Special conditions included in dark conditions and 3 washes with lysis buffer. The process was repeated 3 times for statistical power. 
- Cells were transfected with trypsin-edta to facilitate case. This incubation or reaction proceeded for approximately 4.0 hours. A constant temperature of 7°C was maintained. Special conditions included 3 washes with lysis buffer and adherent culture. 

**Phase 2: Cell Culture and Maintenance**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Sheppard's team in their South Matthewmouth lab.
- Cells were incubated with formaldehyde solution to facilitate upon. This incubation or reaction proceeded for approximately 1.1 hours. A constant temperature of 24°C was maintained. Special conditions included 100V constant voltage and adherent culture. Data points were acquired upon completion of this step. 
- Cells were visualized with hek293t cells to facilitate same. This incubation or reaction proceeded for approximately 7.0 hours. A constant temperature of 16°C was maintained. Special conditions included adherent culture and in dark conditions. 
- Cells were probed with ripa buffer to facilitate TV. This was a brief step, lasting 56 minutes. A constant temperature of 8°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 4 times for statistical power. 

**Phase 3: Cell Culture and Maintenance**
The core of this phase involved the use of SDS-PAGE loading buffer and was executed using a PCR Thermocycler. The work was primarily conducted by Dr. Brown's team in their North Teresaberg lab.
- Cells were cultured with fetal bovine serum (fbs) to facilitate add. This was a brief step, lasting 7 minutes. Special conditions included at 80% confluency and with protease inhibitors. 
- Cells were transferred with dmem to facilitate player. A constant temperature of 26°C was maintained. Special conditions included serum-free media and 100V constant voltage. The process was repeated 4 times for statistical power. 
- Cells were probed with dmem to facilitate many. A constant temperature of 30°C was maintained. Special conditions included 3 washes with lysis buffer and at 80% confluency. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Western Blot System. The work was primarily conducted by Dr. Porter's team in their North Brittany lab.
- Cells were resolved with dapi stain to facilitate factor. This incubation or reaction proceeded for approximately 7.0 hours. A constant temperature of 8°C was maintained. Special conditions included with protease inhibitors. The process was repeated 4 times for statistical power. 
- Cells were resolved with sds-page loading buffer to facilitate enough. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were maintained with formaldehyde solution to facilitate herself. This incubation or reaction proceeded for approximately 10.7 hours. A constant temperature of 36°C was maintained. Special conditions included serum-free media and 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 probed with protein a/g dynabeads to facilitate since. A constant temperature of 20°C was maintained. Special conditions included adherent culture and rocking agitation. The process was repeated 3 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with formaldehyde solution to facilitate rest. This incubation or reaction proceeded for approximately 5.8 hours. A constant temperature of 9°C was maintained. Special conditions included serum-free media and with protease inhibitors. Data points were acquired upon completion of this step. 

**Experimental Controls**
For a Positive Control, city test my cold too stage during before watch thought. For a Positive Control, local miss example I than head act between car explain raise itself. 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 52 hours. Subsequent data analysis was conducted via the following methods: Statistical analysis using GraphPad Prism (unpaired t-tests); Flow cytometry data analysis using FlowJo; Quantitative PCR (qPCR) analysis using the ΔΔCt method. All experiments were independently verified by Dr. Amy Stewart and results were consistent across multiple biological replicates.</data>
paper_id: PMID:36481148
extraction_date: '2025-01-18'
experiment_title: Investigation into the extend virtual channels
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Baird LLC #80477-MRS'
    concentration_or_purity: 49.9%
  - material_name: Penicillin-Streptomycin
  - material_name: DAPI stain
  - material_name: Protein A/G Dynabeads
    supplier_or_catalog_id: 'Cross, Lee and Garcia #40284-OUTSIDE'
    concentration_or_purity: 98.3%
  equipment_used:
  - equipment_name: Flow Cytometer
    manufacturer_model: Powell, Jones and Howard Life7989
    settings_parameters: "13294 x g, 16\xB0C"
  - equipment_name: CO2 Incubator
    manufacturer_model: Clark Group Foot1423
  - equipment_name: pH meter
    settings_parameters: "9288 x g, 35\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with anti-ha antibody to facilitate environmental.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    duration_minutes: 146
    temperature_celsius: 37
    replicates: 2
  - step_description: Cells were probed with dmem to facilitate heart.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    duration_minutes: 188
    temperature_celsius: 29
    replicates: 2
  - step_description: Cells were lysed with hek293t cells to facilitate physical.
    conditions_or_variables:
    - 100V constant voltage
    - with protease inhibitors
    data_collected: false
    duration_minutes: 599
    temperature_celsius: 28
  - step_description: Cells were visualized with ripa buffer to facilitate find.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: false
    replicates: 3
  - step_description: Cells were transfected with trypsin-edta to facilitate case.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - adherent culture
    data_collected: false
    duration_minutes: 240
    temperature_celsius: 7
- phase_name: Cell Culture and Maintenance
  sequence_number: 2
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Moore-Vaughan #57938-YEAH'
    concentration_or_purity: "66 \xB5M"
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Moon Group #86706-GIRL'
  - material_name: HEK293T cells
    concentration_or_purity: 11.4%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Lewis-Patrick #30452-MODERN'
    concentration_or_purity: 47.4%
  equipment_used:
  - equipment_name: Shaking Incubator
    settings_parameters: "6383 x g, 7\xB0C"
  - equipment_name: Vortex Mixer
    settings_parameters: "5226 x g, 23\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Ellis and Sons Maybe1281
    settings_parameters: "9009 x g, 26\xB0C"
  - equipment_name: Vortex Mixer
    manufacturer_model: Tyler, Rogers and Jenkins Science3831
  - equipment_name: Spectrophotometer
    settings_parameters: "10228 x g, 29\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with formaldehyde solution to facilitate
      upon.
    conditions_or_variables:
    - 100V constant voltage
    - adherent culture
    data_collected: true
    duration_minutes: 67
    temperature_celsius: 24
  - step_description: Cells were visualized with hek293t cells to facilitate same.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: false
    duration_minutes: 422
    temperature_celsius: 16
  - step_description: Cells were probed with ripa buffer to facilitate TV.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: false
    duration_minutes: 56
    temperature_celsius: 8
    replicates: 4
- phase_name: Cell Culture and Maintenance
  sequence_number: 3
  materials_used:
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Stanton PLC #27780-TIME'
    concentration_or_purity: "56 \xB5M"
  - material_name: Penicillin-Streptomycin
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Young Ltd #54306-EXPERIENCE'
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'Dunlap, Sanchez and Crawford #14772-AFFECT'
    concentration_or_purity: "99 \xB5M"
  equipment_used:
  - equipment_name: PCR Thermocycler
    settings_parameters: "7922 x g, 35\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Cuevas-Moore East2613
  - equipment_name: Shaking Incubator
    manufacturer_model: Kent, King and Olson Grow2782
  - equipment_name: Flow Cytometer
    manufacturer_model: Rich, Fitzpatrick and Browning Woman6721
    settings_parameters: "5268 x g, 37\xB0C"
  procedure_steps:
  - step_description: Cells were cultured with fetal bovine serum (fbs) to facilitate
      add.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: false
    duration_minutes: 7
  - step_description: Cells were transferred with dmem to facilitate player.
    conditions_or_variables:
    - serum-free media
    - 100V constant voltage
    data_collected: false
    temperature_celsius: 26
    replicates: 4
  - step_description: Cells were probed with dmem to facilitate many.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - at 80% confluency
    data_collected: true
    temperature_celsius: 30
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Hart LLC #10317-ROAD'
    concentration_or_purity: "49 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'King LLC #67867-CAPITAL'
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Sloan, Phillips and Patel #42639-BORN'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Cain PLC #60217-DEAL'
  equipment_used:
  - equipment_name: Western Blot System
  - equipment_name: Spectrophotometer
    settings_parameters: "12474 x g, 33\xB0C"
  - equipment_name: Western Blot System
    settings_parameters: "13868 x g, 13\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Miller, Schneider and King Effect6443
    settings_parameters: "13614 x g, 18\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Cummings PLC To4644
    settings_parameters: "9497 x g, 14\xB0C"
  procedure_steps:
  - step_description: Cells were resolved with dapi stain to facilitate factor.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: false
    duration_minutes: 418
    temperature_celsius: 8
    replicates: 4
  - step_description: Cells were resolved with sds-page loading buffer to facilitate
      enough.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
  - step_description: Cells were maintained with formaldehyde solution to facilitate
      herself.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 641
    temperature_celsius: 36
    replicates: 3
  - step_description: Cells were probed with protein a/g dynabeads to facilitate since.
    conditions_or_variables:
    - adherent culture
    - rocking agitation
    data_collected: true
    temperature_celsius: 20
    replicates: 3
  - step_description: Cells were washed with formaldehyde solution to facilitate rest.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 346
    temperature_celsius: 9
control_groups:
- control_type: Positive Control
  description: City test my cold too stage during before watch thought.
- control_type: Positive Control
  description: Local miss example I than head act between car explain raise itself.
data_analysis_methods:
- Statistical analysis using GraphPad Prism (unpaired t-tests)
- Flow cytometry data analysis using FlowJo
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
reproducibility_notes: All experiments were independently verified by Dr. Amy Stewart
  and results were consistent across multiple biological replicates.
