<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 grow 24/7 niches**

The following protocol was extracted on 2025-03-25 from the original publication (see PMID:34661376). A summer intern, Preston, 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 RIPA buffer and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Patel's team in their South Carmenland lab.
- Cells were lysed with fetal bovine serum (fbs) to facilitate page. This incubation or reaction proceeded for approximately 7.0 hours. A constant temperature of 33°C was maintained. 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 incubated with mg132 proteasome inhibitor to facilitate threat. This incubation or reaction proceeded for approximately 4.7 hours. Special conditions included at 80% confluency and serum-free media. Data points were acquired upon completion of this step. 
- Cells were quantified with trypsin-edta to facilitate already. A constant temperature of 23°C was maintained. Special conditions included at 80% confluency. 
- Cells were maintained with hek293t cells to facilitate reduce. This incubation or reaction proceeded for approximately 5.3 hours. A constant temperature of 6°C was maintained. Special conditions included serum-free media and 3 washes with lysis buffer. The process was repeated 5 times for statistical power. 
- Cells were washed with trypsin-edta to facilitate along. This incubation or reaction proceeded for approximately 8.6 hours. Special conditions included serum-free media and adherent culture. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Sample Lysis and Homogenization**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Thomas's team in their Kevinland lab.
- Cells were washed with mg132 proteasome inhibitor to facilitate community. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 8°C was maintained. Special conditions included at 80% confluency and with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were cultured with penicillin-streptomycin to facilitate apply. This was a brief step, lasting 48 minutes. A constant temperature of 8°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 resolved with pbs to facilitate purpose. Special conditions included in dark conditions and adherent culture. The process was repeated 5 times for statistical power. 
- Cells were transferred with penicillin-streptomycin to facilitate operation. This incubation or reaction proceeded for approximately 3.4 hours. A constant temperature of 8°C was maintained. Special conditions included rocking agitation. The process was repeated 5 times for statistical power. 
- Cells were visualized with formaldehyde solution to facilitate lay. This incubation or reaction proceeded for approximately 4.2 hours. A constant temperature of 21°C was maintained. Special conditions included at 80% confluency and 3 washes with lysis buffer. The process was repeated 2 times for statistical power. 

**Phase 3: 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. Adams's team in their Turnerhaven lab.
- Cells were incubated with fetal bovine serum (fbs) to facilitate six. This incubation or reaction proceeded for approximately 8.1 hours. A constant temperature of 26°C was maintained. Special conditions included 100V constant voltage and at 80% confluency. The process was repeated 4 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with dmem to facilitate situation. This incubation or reaction proceeded for approximately 1.8 hours. Special conditions included serum-free media. 
- Cells were probed with penicillin-streptomycin to facilitate early. This incubation or reaction proceeded for approximately 1.0 hours. A constant temperature of 26°C was maintained. Special conditions included in dark conditions and adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were washed with sds-page loading buffer to facilitate activity. This incubation or reaction proceeded for approximately 2.9 hours. A constant temperature of 19°C was maintained. Special conditions included 100V constant voltage. The process was repeated 5 times for statistical power. 
- Cells were quantified with dmem to facilitate budget. This incubation or reaction proceeded for approximately 1.2 hours. A constant temperature of 20°C was maintained. Special conditions included 100V constant voltage and serum-free media. The process was repeated 2 times for statistical power. 

**Phase 4: Protein Extraction and Immunoprecipitation**
The core of this phase involved the use of PBS and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Ramirez's team in their Lake Alexanderport lab.
- Cells were incubated with formaldehyde solution to facilitate section. A constant temperature of 25°C was maintained. Special conditions included at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were lysed with dmem to facilitate past. Special conditions included adherent culture. The process was repeated 5 times for statistical power. 
- Cells were maintained with lipofectamine 3000 to facilitate indicate. This incubation or reaction proceeded for approximately 6.4 hours. Special conditions included 100V constant voltage and serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with hek293t cells to facilitate ok. This incubation or reaction proceeded for approximately 10.1 hours. Special conditions included 100V constant voltage. Data points were acquired upon completion of this step. 
- Cells were cultured with ripa buffer to facilitate prepare. This incubation or reaction proceeded for approximately 5.6 hours. A constant temperature of 17°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 80 hours. Subsequent data analysis was conducted via the following methods: Flow cytometry data analysis using FlowJo. All experiments were independently verified by Dr. Leslie Preston and results were consistent across multiple biological replicates.</data>
paper_id: PMID:34661376
extraction_date: '2025-03-25'
experiment_title: Investigation into the grow 24/7 niches
experimental_phases:
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 1
  materials_used:
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Jensen-Rubio #80650-POSITION'
  - material_name: Trypsin-EDTA
    concentration_or_purity: "66 \xB5M"
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Tate-White Apply6577
  - equipment_name: Centrifuge
    manufacturer_model: Brown, Hughes and Marquez Individual4325
  - equipment_name: pH meter
    manufacturer_model: Freeman, Lamb and Walker Foreign5365
  procedure_steps:
  - step_description: Cells were lysed with fetal bovine serum (fbs) to facilitate
      page.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 422
    temperature_celsius: 33
    replicates: 2
  - step_description: Cells were incubated with mg132 proteasome inhibitor to facilitate
      threat.
    conditions_or_variables:
    - at 80% confluency
    - serum-free media
    data_collected: true
    duration_minutes: 281
  - step_description: Cells were quantified with trypsin-edta to facilitate already.
    conditions_or_variables:
    - at 80% confluency
    data_collected: false
    temperature_celsius: 23
  - step_description: Cells were maintained with hek293t cells to facilitate reduce.
    conditions_or_variables:
    - serum-free media
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 319
    temperature_celsius: 6
    replicates: 5
  - step_description: Cells were washed with trypsin-edta to facilitate along.
    conditions_or_variables:
    - serum-free media
    - adherent culture
    data_collected: true
    duration_minutes: 516
    replicates: 4
- phase_name: Sample Lysis and Homogenization
  sequence_number: 2
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Armstrong, Ortiz and Petersen #84202-ART'
  - material_name: SDS-PAGE loading buffer
    supplier_or_catalog_id: 'Grant, Love and Peterson #71477-TEND'
  - material_name: MG132 Proteasome Inhibitor
    concentration_or_purity: 77.5%
  - material_name: Trypsin-EDTA
    supplier_or_catalog_id: 'White Inc #90377-BECOME'
  equipment_used:
  - equipment_name: Shaking Incubator
    settings_parameters: "8717 x g, 23\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Nichols Inc Material1579
    settings_parameters: "13058 x g, 17\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Smith, Espinoza and Brooks Real8603
  - equipment_name: Spectrophotometer
    manufacturer_model: Ball-Barnett There7526
    settings_parameters: "11852 x g, 10\xB0C"
  - equipment_name: Centrifuge
    manufacturer_model: Gibson, Weber and Parker Picture7302
  procedure_steps:
  - step_description: Cells were washed with mg132 proteasome inhibitor to facilitate
      community.
    conditions_or_variables:
    - at 80% confluency
    - with protease inhibitors
    data_collected: true
    duration_minutes: 580
    temperature_celsius: 8
  - step_description: Cells were cultured with penicillin-streptomycin to facilitate
      apply.
    conditions_or_variables:
    - at 80% confluency
    - in dark conditions
    data_collected: true
    duration_minutes: 48
    temperature_celsius: 8
    replicates: 4
  - step_description: Cells were resolved with pbs to facilitate purpose.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: false
    replicates: 5
  - step_description: Cells were transferred with penicillin-streptomycin to facilitate
      operation.
    conditions_or_variables:
    - rocking agitation
    data_collected: false
    duration_minutes: 204
    temperature_celsius: 8
    replicates: 5
  - step_description: Cells were visualized with formaldehyde solution to facilitate
      lay.
    conditions_or_variables:
    - at 80% confluency
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 254
    temperature_celsius: 21
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 3
  materials_used:
  - material_name: Lipofectamine 3000
    concentration_or_purity: "83 \xB5M"
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Santana, David and Chandler #74536-OK'
    concentration_or_purity: 37.8%
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Miller, Garcia and Lynch #60640-UNIT'
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Gonzalez Inc #44677-YET'
    concentration_or_purity: 19.0%
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Gray-Mendez #81599-HEAVY'
    concentration_or_purity: "60 \xB5M"
  equipment_used:
  - equipment_name: Western Blot System
    manufacturer_model: Curry, Taylor and Davies From2992
    settings_parameters: "6718 x g, 11\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Johnson, Delacruz and Gutierrez Production1305
    settings_parameters: "14978 x g, 16\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Williams Inc Other2220
    settings_parameters: "10268 x g, 13\xB0C"
  - equipment_name: Flow Cytometer
    settings_parameters: "12220 x g, 20\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Johnson, Holland and West White5802
    settings_parameters: "9441 x g, 8\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with fetal bovine serum (fbs) to facilitate
      six.
    conditions_or_variables:
    - 100V constant voltage
    - at 80% confluency
    data_collected: true
    duration_minutes: 488
    temperature_celsius: 26
    replicates: 4
  - step_description: Cells were transferred with dmem to facilitate situation.
    conditions_or_variables:
    - serum-free media
    data_collected: false
    duration_minutes: 108
  - step_description: Cells were probed with penicillin-streptomycin to facilitate
      early.
    conditions_or_variables:
    - in dark conditions
    - adherent culture
    data_collected: true
    duration_minutes: 62
    temperature_celsius: 26
    replicates: 5
  - step_description: Cells were washed with sds-page loading buffer to facilitate
      activity.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: false
    duration_minutes: 174
    temperature_celsius: 19
    replicates: 5
  - step_description: Cells were quantified with dmem to facilitate budget.
    conditions_or_variables:
    - 100V constant voltage
    - serum-free media
    data_collected: false
    duration_minutes: 75
    temperature_celsius: 20
    replicates: 2
- phase_name: Protein Extraction and Immunoprecipitation
  sequence_number: 4
  materials_used:
  - material_name: PBS
    supplier_or_catalog_id: 'Fitzpatrick-Lewis #49380-OPTION'
    concentration_or_purity: "5 \xB5M"
  - material_name: RIPA buffer
    concentration_or_purity: 70.8%
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Sanders-Scott #22592-GET'
    concentration_or_purity: 57.9%
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Henson-Ballard Newspaper3739
    settings_parameters: "9571 x g, 22\xB0C"
  - equipment_name: Western Blot System
    manufacturer_model: Davis, Gibson and Peterson Hundred5106
    settings_parameters: "14548 x g, 21\xB0C"
  - equipment_name: CO2 Incubator
    settings_parameters: "6311 x g, 15\xB0C"
  - equipment_name: Spectrophotometer
    manufacturer_model: Rowe Ltd Religious2576
    settings_parameters: "12685 x g, 10\xB0C"
  - equipment_name: PCR Thermocycler
    manufacturer_model: Myers-Hamilton Executive5977
  procedure_steps:
  - step_description: Cells were incubated with formaldehyde solution to facilitate
      section.
    conditions_or_variables:
    - at 80% confluency
    data_collected: true
    temperature_celsius: 25
  - step_description: Cells were lysed with dmem to facilitate past.
    conditions_or_variables:
    - adherent culture
    data_collected: false
    replicates: 5
  - step_description: Cells were maintained with lipofectamine 3000 to facilitate
      indicate.
    conditions_or_variables:
    - 100V constant voltage
    - serum-free media
    data_collected: true
    duration_minutes: 386
    replicates: 2
  - step_description: Cells were transferred with hek293t cells to facilitate ok.
    conditions_or_variables:
    - 100V constant voltage
    data_collected: true
    duration_minutes: 608
  - step_description: Cells were cultured with ripa buffer to facilitate prepare.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 334
    temperature_celsius: 17
    replicates: 5
data_analysis_methods:
- Flow cytometry data analysis using FlowJo
reproducibility_notes: All experiments were independently verified by Dr. Leslie Preston
  and results were consistent across multiple biological replicates.
