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]
N/A.

[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
1. 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
2. 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
3. Pipette 20 mL of MesoEndo cell growth medium in a T-75 cell culture flask (Fisher Scientific, Hampton, NH.).
SECTION: Huvec Cell Seeding
4. 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
5. Gently rock the flask for 10 seconds to evenly distribute the cells in the flask.
SECTION: Huvec Cell Seeding
6. Check for cell viability and distribution across the flask using an inverted microscope.
SECTION: Huvec Cell Seeding
7. 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
8. 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
9. 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
10. 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
11. 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
12. 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
13. 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
14. 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
15. 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
16. 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
17. 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
18. 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
18.1. Add the media slowly to prevent the formation of air bubbles inside the slide.
SECTION: Setting up the Ibidi Pump system for ESS
19. 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
20. 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
21. 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
69. 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
22. 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
22.2. Under slide selection, select without any slide.



SECTION: Setting up the Ibidi Pump system for ESS
22.1. Under the fluidic unit set-up, select the appropriate perfusion set.
SECTION: Setting up the Ibidi Pump system for ESS
23. 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
24. 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
24.1. Shear stress – 10 dyn/cm2.
SECTION: Setting up the Ibidi Pump system for ESS
24.2. 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
24.3. All other parameters are calculated automatically (Figure 4).
SECTION: Setting up the Ibidi Pump system for ESS
24.4. If you are running two experiments simultaneously, set P3 as unidirectional and P3 as oscillatory.
SECTION: Protein Detection Using Immnocytochemistry
25. 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
26. 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
27. 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
28. 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
29. 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
30. The following steps must be performed in a dark environment to protect the cells and secondary antibodies from light. 
SECTION: Protein Detection Using Immnocytochemistry
30.1. 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
30.2. 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
30.3. 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
31. 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
32. 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
33. 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
34. 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
35. 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
36. 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
37. Add 350 µL of 70% ethanol to the homogenized cell lysate and mix by pippeting up and down.
SECTION: mRNA Extraction and qRTPCR
38. 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
38.1. Repeat this step for any remaining cell lysate.
SECTION: mRNA Extraction and qRTPCR
39. 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
40. 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
41. 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
41.1. The eluted RNA can be stored at -20°C for a month.
SECTION: mRNA Extraction and qRTPCR
41.2. 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
42. 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
43. 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
43.1. Step 1: 25 °C for 10 minutes.
SECTION: mRNA Extraction and qRTPCR
43.2. Step 2: 37°C for 120 minutes.
SECTION: mRNA Extraction and qRTPCR
43.3. Step 3: 85°C for 5 minutes.
SECTION: mRNA Extraction and qRTPCR
43.4. Step 4: 4°C.
SECTION: mRNA Extraction and qRTPCR
44. 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
45. 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
45.1. One cycle at 50°C for 2 minutes.
SECTION: mRNA Extraction and qRTPCR
45.2. One cycle at 90°C for 20 seconds.
SECTION: mRNA Extraction and qRTPCR
45.3. 40 cycles at 95°C for 1 second, followed by 60°C for 20 seconds.
SECTION: mRNA Extraction and qRTPCR
46. Determine gene expression using the relative standard curve, comparative threshold cycle method, or other methods[1].