TITLE: Quantification of Proteins and Genes Associated with Endothelial Cell Function after Different Shear Stress Intensities in vitro
AUTHORS: Daniel Conde, Mario Garcia, Manuel Gomez, Alvaro Gurovich

[DESCRIPTION]
This protocol describes the steps necessary to culture human umbilical vein endothelial cells (HUVEC), subculture the cells into Ibidi slides, induce endothelial shear stress (ESS) using the Ibidi Pump System, and detect proteins and gene expression associated with endothelial function using immunocytochemistry and qRT-PCR respectively.




[BEFORE_START]
Make sure that the appropriate ethics committee has approved the protocol.

[GUIDELINES]
Use only 70% ethanol as a cleaning agent.

Avoid repeated freeze-thaw cycles.

Avoid exposing fluorescent secondary antibodies or stained slides to direct sunlight.


Figures

 
 
 
 
Tables

     Component      Volume (µL)        10X RT Buffer      2.0        25X dNTP Mix (100 mM)      0.8        10 X RT Randon Primers      2.0        Multiscribe Reverse Transcriptase      1.0        Nuclease-free Water      4.2        Total for One Reaction      10.0   

     Component      Volume (µL)        TaqMan Fast Advanced Master Mix (2X)      10        TaqMan Assay (20X)      1.0        Nuclease-free Water      7.0        Total for One Reaction      18.0   


[STEPS]
SECTION: Huvec Cell Seeding
15. Notes: This step aims to increase the number of HUVEC in a T-75 flask before seeding them in an Ibidi µ-Slide I Luer with a channel height of 0.2 mm. The following procedures must be performed inside a biosafety cabinet with HEPA-filtered laminar flow. To avoid contamination inside the biosafety cabinet, spray all materials with 70% ethanol before placing them inside.
SECTION: Huvec Cell Seeding
16. Place the MesoEndo cell growth medium (Cell Applications, San Diego, CA), and the frozen vial of HUVEC cells (Cell Applications, San Diego, CA.) in a water bath set at 37°C and monitor the cells closely. Once there is only minimal ice, remove the cells and the media and place them inside the biosafety cabinet.
SECTION: Huvec Cell Seeding
17. Pipette 20 mL of MesoEndo cell growth medium in a T-75 cell culture flask (Fisher Scientific, Hampton, NH.).
SECTION: Huvec Cell Seeding
18. Resuspend the cells inside the vial by slowly pipetting up and down five times. Pipette the cells into the T-75 cell culture flask containing the MesoEndo cell growth medium.
SECTION: Huvec Cell Seeding
19. Gently rock the flask for 10 seconds to evenly distribute the cells in the flask.
SECTION: Huvec Cell Seeding
20. Check for cell viability and distribution across the flask using an inverted microscope.
SECTION: Huvec Cell Seeding
21. Incubate the cells overnight at 37°C with 5% carbon dioxide (CO2). Check for cell adhesion to the flask, and change the cell growth medium to remove traces of dimethyl sulfoxide (DMSO) used during cell cryopreservation. Use 20 mL of growth medium in every cell growth media change.
SECTION: Huvec Cell Seeding
22. After the overnight cell growth media change, change the cell growth medium every other day until the cells reach 80% confluence.
SECTION: Subculture into Ibidi µ-Slide I Luer
23. IBIDI offers different µ-Slide versions. This paper uses the µ-Slide I Luer with ibiTreat surface modification for better cell adhesion since we apply high ESS in our experiments. Alternatively, untreated slides can be coated with an extracellular matrix (i.e., collagen). The slides and media must be placed inside the incubator overnight to avoid the formation of bubbles inside the slides.
SECTION: Subculture into Ibidi µ-Slide I Luer
24. Remove the cell medium from the flask and wash the cells twice using Hank’s Buffered Saline Solution (HBSS; Cell Applications, San Diego, CA.) or Dulbecco’s Phosphate Buffered Saline (DPBS; Life Technologies, Carlsbad, CA.)
SECTION: Subculture into Ibidi µ-Slide I Luer
25. After the last wash, completely remove HBSS, add 5 mL of Trypsin/EDTA (Cell Applications, San Diego, CA.), rock the flask until all the cells are covered, and immediately remove 4.5 mL of the Trypsin/EDTA from the flask.
SECTION: Subculture into Ibidi µ-Slide I Luer
26. Monitor the trypsinization of the cells under the microscope. Once the cells become round, tap the flask lightly to detach the cells completely.
SECTION: Subculture into Ibidi µ-Slide I Luer
27. Add 5 mL of trypsin neutralizing solution (TNS; Cell Applications, San Diego, CA.) and transfer the cell suspension to a 50 mL conical tube (Fisher Scientific, Hampton, NH.). Rinse the T-75 flask with 5 mL of TNS and transfer it to the 50 mL conical tube containing the cells.
SECTION: Subculture into Ibidi µ-Slide I Luer
28. Centrifuge the conical tube for 5 minutes at 220 x g to pellet the cells. After pelleting the cells, carefully remove the supernatant without disturbing the pellet.
SECTION: Subculture into Ibidi µ-Slide I Luer
29. Loosen the pellet by flicking the tip of the tube, add 2 mL of cell growth media, and carefully resuspend by pipetting up and down.
SECTION: Subculture into Ibidi µ-Slide I Luer
30. Count the cells and make a suspension with a density of 1 x 106 cells/mL. Dilute the cells with cell growth media if necessary.
SECTION: Subculture into Ibidi µ-Slide I Luer
31. Add 150 µL of the cell suspension directly into the channel inlet of an Ibidi µ-Slide I Luer (Ibidi USA, Fitchburg, WI.) using a 200 µL pipette (Figure 1).
SECTION: Subculture into Ibidi µ-Slide I Luer
32. Incubate the slide at 37°C with 5% carbon dioxide (CO2) and change the media daily until the cells reach 80% confluence.
SECTION: Subculture into Ibidi µ-Slide I Luer
32.1. Add the media slowly to prevent the formation of air bubbles inside the slide.
SECTION: Setting up the Ibidi Pump system for ESS
33. The perfusion set and media must be placed inside the incubator to avoid the formation of bubbles during the ESS experiments. Make sure to use the appropriate perfusion set. For this experiment, we used the yellow-green perfusion set.
SECTION: Setting up the Ibidi Pump system for ESS
34. Place two fluidic units inside the incubator. One unit is used to produce the unidirectional flow, and the second unit creates the pulses. Attach the yellow/green perfusion set (Ibidi USA, Fitchburg, WI.) to the fluidic units, ensuring that the tubing with the yellow/green marks is against the fluidic unit (Figure 2).
SECTION: Setting up the Ibidi Pump system for ESS
35. Pinch the line with the plastic clip, add 5 mL of media to each syringe, and connect the two ends of the perfusion set with the plastic connector.
SECTION: Setting up the Ibidi Pump system for ESS
180. In the Flow Control Software (Ibidi USA, Fitchburg, WI.), load the remove air bubbles demo set-up, remove the plastic clip, and press the play button to remove the bubbles.
SECTION: Setting up the Ibidi Pump system for ESS
36. In the Flow Control Software (Ibidi USA, Fitchburg, WI.), load the remove air bubbles demo set-up, remove the plastic clip, and press the play button to remove the bubbles.
SECTION: Setting up the Ibidi Pump system for ESS
36.2. Under slide selection, select without any slide.



SECTION: Setting up the Ibidi Pump system for ESS
36.1. Under the fluidic unit set-up, select the appropriate perfusion set.
SECTION: Setting up the Ibidi Pump system for ESS
37. Once the bubbles are removed, pinch the line, remove the plastic connector, add approximately 50 µL of media to one of the channel inlets of the Ibidi µ-Slide I Luer, and connect the perfusion set (Figure 3.)
SECTION: Setting up the Ibidi Pump system for ESS
38. Set the ESS conditions in the flow parameters tab using the pump control software. Make sure that fluidic unit 1 (P1) is set as unidirectional and fluidic unit 2 (P2) is set as oscillatory.
SECTION: Setting up the Ibidi Pump system for ESS
38.1. Shear stress – 10 dyn/cm2.
SECTION: Setting up the Ibidi Pump system for ESS
38.2. To achieve 60 pulses per minute, set the oscillatory value to 1 (s). For higher pulses, divide 60 over the target pulses per minute, for example 100 pulses per minute 60/100 = 0.6.
SECTION: Setting up the Ibidi Pump system for ESS
38.3. All other parameters are calculated automatically (Figure 4).
SECTION: Setting up the Ibidi Pump system for ESS
38.4. If you are running two experiments simultaneously, set P3 as unidirectional and P3 as oscillatory.
SECTION: Protein Detection Using Immnocytochemistry
39. Notes: All the dilutions for IHC are made using DPBS, and incubations are performed at room temperature unless otherwise indicated. If two proteins are detected simultaneously, primary antibodies must be from different species (e.g., mouse and rabbit) with corresponding secondary antibodies. For this experiment, we targeted the phosphorylated enzyme endothelial nitric oxide synthase (eNOSp; Thermo Scientific, Ward Hill, MA.) and used the fluorescent secondary antibody Alexa fluor 488 (Invitrogen, Carlsbad, CA.).
SECTION: Protein Detection Using Immnocytochemistry
40. Remove all the cell media and wash the cells with 150 µL of DPBS (Life Technologies, Carlsbad, CA.) and add 150 µL of 2% paraformaldehyde (Thermo Scientific, Ward Hill, MA.) to fix the cells and incubate them for 10 minutes at room temperature.
SECTION: Protein Detection Using Immnocytochemistry
41. Remove the paraformaldehyde and wash the cells three times with DPBS. Add 150 µL of 0.1% Triton X-100 (Millipore Sigma, St Louis, MO) and incubate for 10 minutes at room temperature.
SECTION: Protein Detection Using Immnocytochemistry
42. Remove the Triton X-100 and wash the cells two times with DPBS. Add 150 µL of 1% bovine serum albumin (BSA, Fisher Scientific, Waltham, MA.), and incubate overnight at 4°C.
SECTION: Protein Detection Using Immnocytochemistry
43. Remove the BSA, add 150 µL of 1:100 primary antibody diluted in 1% BSA, and incubate for 60 minutes at 37°C.
SECTION: Protein Detection Using Immnocytochemistry
44. The following steps must be performed in a dark environment to protect the cells and secondary antibodies from light. 
SECTION: Protein Detection Using Immnocytochemistry
44.1. Remove the primary antibody and wash the cells three times with DPBS, add 150 µL of 1:1000 secondary antibody, and incubate for 60 minutes. 
SECTION: Protein Detection Using Immnocytochemistry
44.2. Remove the secondary antibody and wash the cells three times with DPBS, add 150 µL of 1:1000 4’,6-diamino-2-phenylindole (DAPI; Thermo Scientific, Ward Hill, MA.) and incubate for 5 minutes. 
SECTION: Protein Detection Using Immnocytochemistry
44.3. Remove DAPI and wash the cells three times with DPBS. Add 150 µL of DPBS and store the cells at 4°C protected from light.
SECTION: Protein Detection Using Immnocytochemistry
45. Visualize the proteins and nucleus using a confocal microscope. The representative images were taken using the Zeiss LSM confocal microscope (Zeiss, White Plains, NY.).
SECTION: mRNA Extraction and qRTPCR
46. Note: A minimum of four Ibidi µ-Slide I Luer must be used for optimal mRNA concentration. The mRNA extraction uses the Qiagen column-based RNeasy Micro Kit (Qiagen, Hilden, Germany) and TaqMan fast advanced components and the StepOnePlus real time System (Applied Biosystems, Waltham, MA.) for gene expression assessment.
SECTION: mRNA Extraction and qRTPCR
47. Remove all the cell media, wash the cells five times with DPBS, and add 100 µL of RLT buffer to lyse the cells.
SECTION: mRNA Extraction and qRTPCR
48. Attach a 1 mL syringe to one of the channel inlets and move the plunger quickly up and down several times to shear out the lysed cells. 
SECTION: mRNA Extraction and qRTPCR
49. Add an additional 100  µL of RLT buffer and shear out any remaining the cells in the slide. In a 1.5 mL microcentrifuge tube mix the 200 µL of cell lysate with 150 µL of RLT buffer to achieve a total volume of 350 µL.
SECTION: mRNA Extraction and qRTPCR
50. Using a new syringe with a 20-gauge needle, pass the entire cell lysate 15 times. Completely homogenize the lysate by vortexing for 30 seconds.
SECTION: mRNA Extraction and qRTPCR
51. Add 350 µL of 70% ethanol to the homogenized cell lysate and mix by pippeting up and down.
SECTION: mRNA Extraction and qRTPCR
52. Bind the total RNA to a MinElute spin colum, by transferring up to 700 µL of the cell lysate. Centrifuge the column at 8,000 x g for 15 seconds and discard the flow through from the collection tube.
SECTION: mRNA Extraction and qRTPCR
52.1. Repeat this step for any remaining cell lysate.
SECTION: mRNA Extraction and qRTPCR
53. To digest DNA, add 350 µL of RW1 buffer, centrifuge at 8,000 x g for 15 seconds and discard the flow through. Add DNase I incubation mix and incubate for 15 minutes. Add 350 µL of RW1 buffer, centrifuge at 8,000 x g for 15 seconds and discard the flow through.
SECTION: mRNA Extraction and qRTPCR
54. To wash the spin column, use a new collection tube. Add 500 µL of RPE buffer and centrifuge at 8,000 x g for 15 seconds. Repeat this step. Add 500 µL of 80% ethanol, centrifuge at 8,000 x g for 2 minutes and discard the flow through and collection tube.
SECTION: mRNA Extraction and qRTPCR
55. To elute the RNA, place the spin column in a new collection tube and centrifuge at >12,000 x g for five minutes and discard the flow through and collection tube. Place the spin column into a 1.5 mL tube, add 14 µL of RNA free water, incubate for 3 minutes and centrifuge at >12,000 x g for one minute.
SECTION: mRNA Extraction and qRTPCR
55.1. The eluted RNA can be stored at -20°C for a month.
SECTION: mRNA Extraction and qRTPCR
55.2. The concentration of RNA can be measured using the absorbance at 260 nm. The ratio of absorbance at 260 nm and 280 nm provides a measurement for the purity of the eluted RNA.
SECTION: mRNA Extraction and qRTPCR
56. Prepare a reverse transcription master mix for up to 2 µg of mRNA to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA.) following the volumes in Table 1.
SECTION: mRNA Extraction and qRTPCR
57. Load 10 µl of the reverse transcription master mix in a PCR plate or PCR tubes. Add 10 µL of the eluted mRNA and load the plate/tubes in a thermocycler. Set the thermocycler reaction as follows:
SECTION: mRNA Extraction and qRTPCR
57.1. Step 1: 25 °C for 10 minutes.
SECTION: mRNA Extraction and qRTPCR
57.2. Step 2: 37°C for 120 minutes.
SECTION: mRNA Extraction and qRTPCR
57.3. Step 3: 85°C for 5 minutes.
SECTION: mRNA Extraction and qRTPCR
57.4. Step 4: 4°C.
SECTION: mRNA Extraction and qRTPCR
58. Prepare a PCR master mix using the TaqMan Fast Advanced Master Mix (Applied Biosystems, Waltham, MA.) following volumes in Table 2. 
SECTION: mRNA Extraction and qRTPCR
59. Load 18 µL of the PCR master mix in a PCR plate or PCR tubes. Add 2 µL of cDNA and assess gene expression on the StepOnePlus PCR system following the following reaction set-up:
SECTION: mRNA Extraction and qRTPCR
59.1. One cycle at 50°C for 2 minutes.
SECTION: mRNA Extraction and qRTPCR
59.2. One cycle at 90°C for 20 seconds.
SECTION: mRNA Extraction and qRTPCR
59.3. 40 cycles at 95°C for 1 second, followed by 60°C for 20 seconds.
SECTION: mRNA Extraction and qRTPCR
60. Determine gene expression using the relative standard curve, comparative threshold cycle method, or other methods[1].
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
1. Notes: Two exercise tests must be performed on a cycle ergometer. The first test determines maximal oxygen uptake (VO2max), maximal heart rate (HRmax), and lactate threshold (La). The second test determines the three exercise intensities for measuring endothelial shear stress (ESS). There must be at least 48 hours between exercise tests. ESS is expressed in dynes/cm2.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
2. Obtain the weight and height of the participants using a standard weighing scale and stadiometer.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
3. Adjust the height of the bike’s seat to allow a 5° to 15° knee flexion.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
4. Have the participant quietly sit on the bike for 10 minutes to ensure sympathetic activity does not alter blood pressure readings.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
6. Measure VO2, HR, and La at the end of each two-minute stage.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
5. Perform the VO2max test following the American Heart Association (AHA) and the American College of Sports Medicine (ACSM) guidelines.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
4.1. Measure the participant’s blood pressure at the end of the 10 minutes and repeat the measurements three times.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
4.2. Average the blood pressure readings.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
5.1. Have the participants complete 2-minute stages until voluntary exhaustion.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
5.2. Increase the workload by 25 Watts after each stage.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
5.3. Increase the workload by 25 Watts after each stage.
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
6.1. VO2 is measured with a metabolic cart (i.e., TrueOne 2400, Parvomeics Inc., Sandy, UT).
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
6.2. HR is measured with a cardiac stress system (i.e., Quinton, Q-Stress Cardiac Stress System, Mortara Instrument, Milwaukee, WI).
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
6.3. La is measured using a micro-sample of blood from the earlobe and an automated blood lactate analysis (e.g., Lactate Plus, Nova, Inc., Boston, MA).
SECTION: Determination of Exercise Intensities for Exercise-Induced Endothelial Shear Stress Measurements
7. At the end of each test, determine three exercise intensities by determining three La curves as follows: Low-intensity (0-2 mmol/L), Moderate-intensity (2-4 mmol/L), and High-intensity (>4 mmol/L).
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
8. Before the start of the exercise test, determine the participant’s hematocrit from 2 earlobe capillary blood samples.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
8.1. Collect the blood using microhematocrit capillary tubes and seal them.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
8.2. Centrifuge and determine hematocrit using a semi-automated hematocrit measuring device (i.e., HemataStat II Hematocrit Analyzer, Separation Technology Inc.).
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
9. Have the participant exercise for 5 minutes at each predetermined intensity. 
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
9.1. Monitor VO2 and HR throughout the exercise session.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
9.2. Assess La at minutes 2 and 4 of each exercise intensity to confirm that the exercise intensity corresponds to the La expected at each intensity.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
10. Measure blood flow patterns in the carotid and/or brachial artery using a 12 to 18 Hz ultrasound transducer (i.e., LA435, Esaote, Firenze, Italy).
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
10.1. Brachial artery – 5 cm proximal to the antecubital fossa. Arm placed on a flat surface, maintaining a ~80° of shoulder abduction and a 35° to 45° flexion.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
10.2. Common carotid artery – lateral aspect of the neck.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
12. Identify a transverse section of the artery and center it.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
11. Adjust the ultrasound settings to 12 Hz frequency and 3 cm depth for the brachial artery and 12 to 18 Hz and 3 cm to 5 cm depth for the common carotid artery.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
13. Rotate the transducer 90° to obtain a longitudinal view of the artery and use a pulse wave Doppler to record blood flow velocity.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
13.1. Ensure an insonation angle below 60°.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
13.2. For the carotid artery place the Doppler 5 to 10 mm distal to the bifurcation.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
13.3. For the blood flow of the internal and external carotid arteries, move the Dopper 5 to 12 mm proximal to the bifurcation.
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
14. Calculate ESS using the Womersley approximation as follows:
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
14.1. ESS = µ X SR
SR = 2K X V/D
µ is blood viscosity 
SR is the shear rate
V is the peak systolic velocity
D is artery diameter
K is the complex factor dependent on the Womersly parameter α
SECTION: Exercise-Induced Endothelial Shear Stress Measurements
14.2. α = (D/2) X (ω/[μ/ρ])1/2
D is artery diameter 
 ω is the angular frequency of flow pulsation (ω = freq X 2π)
 ρ is blood density 
 μ is blood viscosity 
SECTION: Protein Extraction and Western Blot Analysis
61. Add 10 mL of Dulbecco’s Phosphate Buffered Saline (DPBS) in a 15 ml conical tube.
SECTION: Protein Extraction and Western Blot Analysis
61.1. Keep it in ice or refrigerated until use
SECTION: Protein Extraction and Western Blot Analysis
62. Prepare 1 mL lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF).
SECTION: Protein Extraction and Western Blot Analysis
62.1. Add the PMSF immediately before use.
SECTION: Protein Extraction and Western Blot Analysis
62.2. Keep it in ice or refrigerated until use.  
SECTION: Protein Extraction and Western Blot Analysis
63. Pre-cool a syringe, needle, and two 1.5 mL tubes by placing them in ice or the refrigerator (4°) 15 to 20 minutes before starting the extraction.
SECTION: Protein Extraction and Western Blot Analysis
65. Wash the channels 3 times using cold PBS.
SECTION: Protein Extraction and Western Blot Analysis
64. Remove the Ibidi slides from the incubator and immediately place them on ice.
SECTION: Protein Extraction and Western Blot Analysis
64.1. All the following steps are performed with the slides on the ice.
SECTION: Protein Extraction and Western Blot Analysis
65.1. Remove the cell culture media by aspirating it from one of the luers.
SECTION: Protein Extraction and Western Blot Analysis
65.2. Pipette about 100 µL of cold DPBS in one luer and remove from the opposite luer.
SECTION: Protein Extraction and Western Blot Analysis
66. After the third wash, add cold DPBS and keep the slide on ice until lysed.
SECTION: Protein Extraction and Western Blot Analysis
66.1. Lyse only one slide at a time and keep the rest in ice.
SECTION: Protein Extraction and Western Blot Analysis
67. Remove the DPBS from the slide, and using the pre-cool syringe, add 200 µL of cold lysis buffer with PMSF.
SECTION: Protein Extraction and Western Blot Analysis
67.1. Make sure the slide channel is completely covered with lysis buffer.
SECTION: Protein Extraction and Western Blot Analysis
67.2. Remove bubbles by pumping the syringe’s plunger up and down to move the buffer inside the slide back and forth.
SECTION: Protein Extraction and Western Blot Analysis
68. Incubate for 5 minutes.
SECTION: Protein Extraction and Western Blot Analysis
68.1. Keep the slide on ice and the syringe attached to the luer.
SECTION: Protein Extraction and Western Blot Analysis
69. Remove the lysed cells by quickly moving the plunger of the syringe up and down.
SECTION: Protein Extraction and Western Blot Analysis
69.1. Make sure not to squeeze the cell lysate out of the other luer.
SECTION: Protein Extraction and Western Blot Analysis
70. Remove all the lysate from the slide and keep it inside the syringe.
SECTION: Protein Extraction and Western Blot Analysis
71. Remove the DPBS for the next slide, add the lysate from the syringe, and repeat steps 67 - 70.
SECTION: Protein Extraction and Western Blot Analysis
72. Once all the slides have been completed, collect the lysate in the pre-cooled 1.5 mL tube.
SECTION: Protein Extraction and Western Blot Analysis
73. Attach the needle to the syringe and pass the entire lysate through it 15 times.
SECTION: Protein Extraction and Western Blot Analysis
73.1. Do it slowly to avoid theexcessive creation of bubbles.
SECTION: Protein Extraction and Western Blot Analysis
76. Proceed to electrophoresis or freeze the sample.
SECTION: Protein Extraction and Western Blot Analysis
75. Collect the supernatant in the remaining pre-cooled 1.5 mL tube.
SECTION: Protein Extraction and Western Blot Analysis
74. Centrifuge the lyste at 14,000 x g at 4 °C for 10 minutes. 
SECTION: Protein Extraction and Western Blot Analysis
76.1. If used immediately, the sample can be maintained at 4 °C or in ice.
SECTION: Protein Extraction and Western Blot Analysis
76.2. If not used immediately, store the sample at -20 °C for a few days to up to 2 weeks, or at -80 °C for up to 6 months.
SECTION: Protein Extraction and Western Blot Analysis
77. Perform a bicinchoninic acid (BCA) assay or another method to quantify each extracted sample's protein content.
SECTION: Protein Extraction and Western Blot Analysis
78. Prepare 1 mL of 4X Laemly with 1M dithiothreitol (DTT).
SECTION: Protein Extraction and Western Blot Analysis
78.1. Weight 154.25 mg of DTT and add it to the Laemli buffer.
SECTION: Protein Extraction and Western Blot Analysis
79. Dilute the proteins using the 4X Laemly buffer to achieve 30 µg per sample.
SECTION: Protein Extraction and Western Blot Analysis
80. Use a Biorad pre-cast gel and assemble the electrophoresis cassette.
SECTION: Protein Extraction and Western Blot Analysis
80.1. Remove the tape at the bottom of the pre-cast gel before assembly.
SECTION: Protein Extraction and Western Blot Analysis
82. Load 2 µL of Biorad dual color molecular weight marker in the first lane and 30 µL of sample in the remaining lanes.
SECTION: Protein Extraction and Western Blot Analysis
81. Place the cassette in the boat and add enough Tris-glycine sodium dodecyl sulfate (TGS) buffer.
SECTION: Protein Extraction and Western Blot Analysis
81.1. Remove the comb slowly to keep the well walls straight.
SECTION: Protein Extraction and Western Blot Analysis
82.1. Load the proteins slowly to avoid spills of samples to other wells.
SECTION: Protein Extraction and Western Blot Analysis
83. Run the electrophoresis apparatus at 50 V for about 2.5 hours until the proteins run to the bottom of the gel and stop the current.
SECTION: Protein Extraction and Western Blot Analysis
84. Disassemble the cassette, rinse the pre-cast gel with deionized (DI) water, and break the plastic casing to remove the gel. 
SECTION: Protein Extraction and Western Blot Analysis
84.1. Remove one side of the casing, leaving the gel on one side of the casing.
SECTION: Protein Extraction and Western Blot Analysis
85. Open a Biorad transfer pack to create a transfer sandwich.
SECTION: Protein Extraction and Western Blot Analysis
86. Place the gel on top of the membrane.
SECTION: Protein Extraction and Western Blot Analysis
85.1. Remove the contents of the side labeled bottom which contains the membrane, and place iton the bottom piece of the transfer cassette  
SECTION: Protein Extraction and Western Blot Analysis
86.1. Use the roller to remove air bubbles.
SECTION: Protein Extraction and Western Blot Analysis
87. Remove the contents of the side labeled top and place it on top of the gel.
SECTION: Protein Extraction and Western Blot Analysis
87.1. Use the roller to remove air bubbles.
SECTION: Protein Extraction and Western Blot Analysis
88. Close the transfer cassette and place it on the Biorad Trans-Blot Turbo Transfer System.
SECTION: Protein Extraction and Western Blot Analysis
88.1. Run the preset “turbo” and “1 mini TGX”.
SECTION: Protein Extraction and Western Blot Analysis
88.2. The system automatically makes the transfer in 3 minutes.
SECTION: Protein Extraction and Western Blot Analysis
89. When the transfer is completed, remove and open the cassette.
SECTION: Protein Extraction and Western Blot Analysis
90. Place the membrane in an incubation box and add enough LiCor intercept blocking buffer to cover the membrane.
SECTION: Protein Extraction and Western Blot Analysis
89.1. Dispose of the filters from the sandwich and gel.
SECTION: Protein Extraction and Western Blot Analysis
89.2. A successful transfer shows the molecular weight marker in the membrane.
SECTION: Protein Extraction and Western Blot Analysis
90.1. The membrane should be floating in the box.
SECTION: Protein Extraction and Western Blot Analysis
90.2. Remove any air bubbles from the bottom of the membrane by rocking the box.
SECTION: Protein Extraction and Western Blot Analysis
90.3. If bubbles cannot be removed by rocking the box, lift the membrane using forceps and slowly
place it back in the box.
SECTION: Protein Extraction and Western Blot Analysis
91. Place the incubation box on a shaker set at 130 revolutions per minute (RPM) and incubate it for 2 hours at room temperature (22 °C to 25 °C).
SECTION: Protein Extraction and Western Blot Analysis
92. Prepare 20 μL of the primary antibody by diluting it in blocking buffer according to the vendor’s recommendation for western blot.
SECTION: Protein Extraction and Western Blot Analysis
92.1. Abcam’s eNOS primary antibody is recommended as 1:1000.
SECTION: Protein Extraction and Western Blot Analysis
94. Place the incubation box in a shaker set at 130 RPM and incubate overnight at 4 °C.
SECTION: Protein Extraction and Western Blot Analysis
93. After the incubation in blocking buffer, remove it from the incubation box and add the diluted primary antibody.
SECTION: Protein Extraction and Western Blot Analysis
93.1. The blocking buffer can be saved at 4 °C and re-used 3 times.
SECTION: Protein Extraction and Western Blot Analysis
94.1. It can be placed in a cold room if available or inside a refrigerator.
SECTION: Protein Extraction and Western Blot Analysis
95. After overnight incubation, remove the primary antibody and wash the membrane 3 times using Tris-buffered saline (TBS).
SECTION: Protein Extraction and Western Blot Analysis
95.2. The diluted primary antibody can be saved at 4 °C and re-used approximately 2 or 3 times.
SECTION: Protein Extraction and Western Blot Analysis
95.1. Add TBS to the incubation box, place it on the shaker for 10 minutes, and discard the TBS.
SECTION: Protein Extraction and Western Blot Analysis
96. Prepare a fluorescent secondary antibody following the vendor’s recommendation for western blot.
SECTION: Protein Extraction and Western Blot Analysis
96.1. LiCor IRDye 800 fluorescent secondary antibody is recommended at 1:15,000.
SECTION: Protein Extraction and Western Blot Analysis
96.2. The secondary antibody must be from the same species as the primary antibody.
SECTION: Protein Extraction and Western Blot Analysis
98. After incubation, wash the membrane 3 times with TBS.
SECTION: Protein Extraction and Western Blot Analysis
97. Incubate at room temperature for 2 hours, protecting it from light.
SECTION: Protein Extraction and Western Blot Analysis
99. If normalization is required, follow steps 93 - 98, describing the incubation in primary and secondary
antibodies.
SECTION: Protein Extraction and Western Blot Analysis
100. Scan the membrane using a near-infrared fluorescent scanner such as the Li-Cor Odyssey DLx.
SECTION: Protein Extraction and Western Blot Analysis
100.1. The membrane can be saved at 4 °C in TBS.
SECTION: Protein Extraction and Western Blot Analysis
100.2. Alternatively, the membrane can be dried in between two filter papers.