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Protocol

Single-Cell Intracellular Proteome: HeLa Cell Protocol

  • IsoPlexis Applications Team 1
  • 1 - IsoPlexis

Aug 03, 2021

Abstract

Overview of Protocol:

Day 1-3: Cryopreserved HeLa cells are thawed and cultured for 72 hours. 

Day 4: Staining and stimulating of HeLa cells followed by immediate loading onto IsoCode Chip


Introduction

NOTE: This protocol outlines the standard method for thawing and culturing of human HeLa cells only and may not be valid for other species or cell types. 

NOTE: Using stains and protocols other than recommended stain and protocols might result in failed runs. Stains and staining procedures not approved by IsoPlexis will require validation prior to use. Please consider IsoPlexis’ IsoPACE™ program to assist in custom marker and protocol validation. 

NOTE: This protocol focuses on HeLa cell handling and signaling stimulation/repression with hEGF, hIFN-α, and Calyculin A. If you are using a different cell type or have a different drug treatment paradigm that you would like to analyze, please work with your local FAS to plan your experiment. Over 20 cell lines have been tested on the Intracellular Proteome Solution.

Safety Warnings:

Read MSDS documents of all materials prior to use. Laboratory workers should wear standard PPE including disposable gloves, protective eyewear, and laboratory coats. 


Reagents and Equipment

Required Reagents, Consumables and Equipment

IsoCode Kit Components:

  • IsoCode Reagent Box (4°C) 
    • 15 mL Tube A 
    • 15 mL Tube B 
    • 1.5 mL Tube A: Cocktail A in Micro-Tube (Green Cap) 
    • 50 mL Tubes containing Reagents 1, 3, 4, 5, 6, 7, 8 
    • 1 Bag of Disposable Reagent Sippers 
  • IsoCode Chip Set (-20°C) 
    • Boxes of IsoCode Chips (2 per box) to make up 4 chips 
    • 50 mL tube containing reagent 2 
    • Membrane Stain [ordered separately] 
    • Membrane Stain Diluent (DMSO) [part of membrane stain kit]

 


Procedure

Before Getting Started

 1. Important Precautions 

Read MSDS documents of all materials prior to use. 

Working with Biohazardous Reagents

Please refer to your institute’s guidelines and obtain proper training to handle potentially biohazardous samples. It is also strongly recommended that any lab personnel handling human samples should be vaccinated against HBV if the individual does not have sufficient HBV antibody titer. 

Additional precautions need to be taken when working with samples that potentially contain an EID agent: 

  • Laboratory workers should wear standard PPE including disposable gloves, protective eyewear, and laboratory coats.
  • Any procedure or process that cannot be conducted in the designated EID BSC should be performed while wearing gloves, gown, goggles and a fit tested N-95 mask.
  • Work surfaces should be decontaminated on completion of work with appropriate disinfectants. This includes any surface that potentially comes in contact with the specimen (centrifuge, microscope, etc.).
  • All liquid waste produced in the processes must be treated to a final concentration of 10% bleach prior to disposal.

2. Reagents to Be Prepared Before Starting

Additional Reagent to Be Prepared:

CRITICAL: Calyculin A should be aliquoted upon receipt from vendor. 5-10 µL aliquots are recommended and storage is at -20°C for up to 3 months.

Protocol

Chapter 1: Getting Started

Kit Contents:

  • IsoCode Reagent Box (4°C)
    • 15 mL Tube A
    • 15 mL Tube B
    • 1.5 mL Tube A: Cocktail A in Micro-Tube (Green Cap)
    • 50 mL Tubes Containing Reagents 1, 3, 4, 5, 6, 7, 8
    • 1 Bag of Disposable Reagent Sippers
  • IsoCode Chip Set (-20°C)
    • Boxes of IsoCode Chips (2 Per Box) to Make Up 4 Chips
    • 50 mL Tube containing Reagent 2
    • Membrane stain [ordered separately]
    • Membrane stain diluent (DMSO) [part of Membrane stain kit]

Chapter 2: Recovery of Cryopreserved Cells 

Materials Required:

  • Complete RPMI (37°C)
  • Cryopreserved HeLa Cells
  • 15 mL Centrifuge Tube
  • Lo-Bind Microcentrifuge Tube for Cell Count
  • Plate and/or Flask
    • For 2-5 M cells, T75 Flask
    • For 1-2 M cells, T25 Flask
    • For < 1 M cells, 6 Well Plate

All the following steps should take place in a sterile tissue culture hood. Centrifugation steps should be performed at 21°C. 

Methods:

1. Pipette 5 mL of complete RPMI into a 15 mL centrifuge tube, labeled Thawed HeLa. 

2. Using proper PPE, remove cells from liquid nitrogen storage and thaw cells. TIP: Be careful of contamination. 

3. Quickly move vials into a water bath (37°C) to thaw. While thawing, swirl the vial in the water until a single ice crystal remains in the vial. Be sure to prevent (to the best of your ability) any of the water from the water bath from getting under the cap and into the sample. 

4. When the sample is nearly thawed, remove the vial and immediately spray vial with 70% alcohol before bringing into the hood. It is important to allow the alcohol to evaporate before opening the vial.

5. Slowly pipette thawed cells into 5 mL of complete RPMI in 15 mL centrifuge tube, labeled Thawed HeLa. TIP: Insert tip into complete RPMI when pipetting, be careful to not create bubbles. 

6. Take 1 mL of the cell/complete RPMI mixture. 

7. Pipette into original thawed cell vial, rinse inside the vial with the complete RPMI to recover additional thawed cells. TIP: Insert tip into complete RPMI, be careful to not create bubbles. 

8. Draw up cell/complete RPMI mixture and pipette back into the 15 mL centrifuge tube. TIP: Insert tip into complete RPMI and pipette gently up and down. Be careful to not create bubbles

9. Centrifuge cells for 10 minutes at 300 rcf. 

10. After cells are centrifuged, check for cell pellet. 

11. Aspirate supernatant. TIP: Be careful not to aspirate cell pellet.

  • Use pipette to remove last bit of supernatant.

12. Resuspend cell pellet in 1 mL of fresh complete RPMI. 

  • Mix well to resuspend. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles.
  • Take 10 µL aliquot of your cells and transfer to a Lo-Bind Microcentrifuge Tube for cell counting. CRITICAL: See Appendix D1 for cell counting instructions. 

13. Slowly add additional complete RPMI to a final concentration of 1 x 105 cells/mL. 

14. Mix with serological pipette. TIP: Gently pipet up and down 3-5 times, be careful to not create bubbles. 

15. Transfer cell suspension to flask or plate. TIP: Slowly pipette down the side of the flask as to not create bubbles. 

16. Spread out cell suspension by rocking flask carefully to fully cover the bottom of the flask. TIP: Be careful to not make bubbles. 

17. Move to incubator for 72-hour recovery at 37°C, 5% CO2 to ensure that cells are fully recovered from freezing. Cells should be growing in log phase and at ~70-80% confluency before plating for treatment. 

Chapter 3: Lifting and Staining Recovered Cells 

Materials Required:

  • Complete RPMI (37°C) 
  • TrypLE Express Enzyme 
  • Membrane Stain 
  • PBS 
  • 15 mL Centrifuge Tube 
  • Recovered Cells from Chapter 2 
  • 2 x Lo-Bind Microcentrifuge Tube

All the following steps should take place in a sterile tissue culture hood. Centrifugation steps should be performed at 21°C. 

CRITICAL: Before starting this section, ensure that you have enough time to complete Chapters 3 – 6 as you cannot stop. This is due to the transient nature of protein phosphorylation. Estimated time is 90-120 minutes depending on the number of samples and familiarity with the protocol. It is also critical to begin chip and lysis buffer thawing (Reagent 2) at this time so that run can be initiated immediately after cell stimulation and chip loading. Reagent A should be prepared, and all additional reagent tubes should be put on the IsoLight before proceeding. Refer to Chapter 4 for guidance on these steps. 

Methods:

1. Observe flask or plate under light microscope and ensure cells are well-adhered and between 50 and 80% confluent. If so, move to Step 2 and if not, continue culturing until desired confluency is reached. 

2. Aspirate supernatant and any cells in suspension. The supernatant and cells in suspension can be discarded. TIP: Be careful to not dislodge any cells adhered to the plate. 

3. Add PBS to flask or plate in the following volumes: 

  • For T75 Flask: 3 mL 
  • For T25 Flask: 2 mL 
  • For 6 Well Plate: 1 mL 

4. Gently rinse the cells by rocking flask or plate back and forth. 

5. Aspirate PBS being careful not to dislodge any cells adhered to the plate. 

6. Add TrypLE Express Enzyme to the flask or plate in the following volumes: 

  • For T75 Flask: 5 mL 
  • For T25 Flask: 3 mL 
  • For 6 Well Plate: 1 mL 

TIP: Gently rock flask or plate to ensure TrypLE is distributed evenly across entire surface area. 

7. Place flask or plate in incubator for 5 minutes. 

8. After incubation, check under microscope to determine if cells are detached. If so, proceed to next step, if not place in incubator and continue to check every 90 seconds. 

9. Rinse flask or plate with media using the following volumes: 

  • For T75 Flask: 10 mL
  • For T25 Flask: 6 mL 
  • For 6 Well Plate: 2 mL 

TIP: Dispense media from top of plate/flask while angling the plate or flask so media washes down all cells – while angled down, you can tilt flask side to side to ensure all cells are washed down with media

10. Transfer cell suspension to 15 mL centrifuge tube. 

11. Centrifuge cells for 10 minutes at 300 rcf. 

12. After cells are centrifuged, check for cell pellet.

13. Aspirate supernatant. TIP: Be careful not to aspirate cell pellet.

  • Use pipette to remove last bit of supernatant. 

14. Resuspend pellet in 1 mL of PBS. 

15. Centrifuge cells for 10 minutes at 300 rcf. 

TIP: While cells are centrifuging, membrane stain can be prepared. 

16. Prepare membrane stain stock. 

  • Thaw tube of membrane stain diluent (DMSO) at room temperature.
  • Spin tubes of membrane stain and membrane stain diluent (DMSO) in a mini centrifuge for 10 seconds to collect the contents at the bottom of the tubes.
  • Add 20 µL of membrane stain diluent (DMSO) directly to the tube of membrane stain. Pipette up and down 15 times gently to resuspend. 

CRITICAL: Membrane stain must be prepared fresh. Discard remaining stain – do not store. 

17. Prepare stain master mix by diluting 2 µL of membrane stain into 1 mL of 1X PBS in a Lo-Bind microcentrifuge tube (1:500 final dilution). With the same pipette tip, pipette up and down 10 times to ensure all membrane stain has been released. Depending on sample number and cell count, additional tubes of stain master mix may need to be prepared. CRITICAL: Failure to follow these steps will negatively impact cell counts. 

  • With a P1000 set to 500 µL, gently pipette the stain master mix up and down 15 times.
  • Gently vortex the stain master mix for 5 seconds. 
  • Ensure master mix is mixed well before adding stain to cells. 

18. After cells are centrifuged, check for cell pellet. 

19. Aspirate supernatant. TIP: Be careful not to aspirate cell pellet. 

  • Use pipette to remove last bit of supernatant. 

20. Resuspend pellet in 1 mL of well-mixed stain master mix. 

21. Place in incubator for 12 minutes. 

22. Quench stain with 5 mL of RPMI and take 10 µL aliquot of your cells and transfer to a Lo-Bind Microcentrifuge Tube for cell counting. If running stimulated and unstimulated cells, pipette half of the cell suspension into a 2nd 15 mL centrifuge tube. CRITICAL: See Appendix D1 for cell counting instructions. 

23. Centrifuge cells for 10 minutes at 300 rcf. While cells are centrifuging, use hemocytometer to count cells. CRITICAL: See Appendix D1 for cell counting instructions. 

CRITICAL: During this time, refer to Table 10 in Chapter 5 to determine volume of PBS required to resuspend stimulated cell pellet. 

24. After cells are centrifuged, check for cell pellet. 

25. Aspirate supernatant. TIP: Be careful not to aspirate cell pellet. 

  • Use pipette to remove last bit of supernatant. 

26. Using cell count from Step 23, resuspend cell pellets in PBS. Unstimulated cell suspension should be at 1x106 cells/mL. Stimulated cell suspension should be at volume determined based on Table 10 (Chapter 5). 

Chapter 4: Chip and Reagent Thaw 

Materials Required:

  • IsoCode Chips in Vacuum Sealed Bag (-20°C) 
  • 50 mL tube containing reagent 2 (-20°C) 

Methods:

 1. Take vacuum sealed bag containing IsoCode chips from -20°C. CRITICAL: Chips must stay sealed until Chip Loading (Chapter 6). 

2. Place on bench to thaw at ambient temperature 30 - 60 minutes prior to use. 

3. Take 50mL tube containing reagent 2 from -20°C to thaw at ambient temperature 90-120 minutes prior to use. CRITICAL: Ensure reagent 2 is completely thawed prior to Chip Loading (Chapter 6). 

While chips and samples thaw, prepare liquid reagents and attach all reagent tubes to IsoLight. Refer to the IsoLight System Guide for detailed instructions. 

Chapter 5: Cell Stimulation

Materials Required:

  • PBS 
  • hEGF 
  • hIFN-α 
  • Calyculin A 
  • Protease Phosphatase Inhibitor Cocktail 
  • Cell suspension from Chapter 3 

All the following steps should take place in a sterile tissue culture hood. Centrifugation steps should be performed at 21°C. 

Methods:

CRITICAL: Protein phosphorylation is a highly transient process making stimulation time critical. Before adding any of the reagents mentioned in this section, be sure to have IsoCode chips thawed, reagents on IsoLight and data upload location set. 

1. Retrieve pre-made single use aliquots of hEGF, hIFN-α, and Calyculin A from -20°C. 

2. Thaw reagent tubes at ambient temperature. Spin down in a mini centrifuge for 10 seconds to collect the contents at the bottom of the tubes. TIP: Ensure that contents are all in the bottom of the vial. 

  • Dilute aliquot of hIFN-α 1:100.
  • Dilute aliquot of Calyculin A 1:10. 
  • hEGF will be used at stock concentration (no dilution necessary). 

3. Using cell count from Chapter 3 Step 23, determine the amount of each stimulant required. See Table 10 for example calculations. 

  • hIFN-α will be used at a 100X dilution in a 1:20 ratio (final concentration 50 ng/mL).
  • Calyculin A will be used at a 10X dilution in a 1:50 ratio (final concentration 50 nM). 
  • hEGF will be used at stock concentration at a 1:20 ratio (final concentration 5 µg/mL) 

TIP: 100,000 cells would require a final volume of 100 µL for 1 x 106 cells/mL. Given the ratios mentioned, 5 µL stock hEGF would be needed, 5 µL 100X diluted hIFN-α would be needed and 2 µL 10X diluted Calyculin A would be needed – all added to 88 µL of cell suspension in PBS. 

4. Add stimulants to stimulated cell suspension and protease phosphatase inhibitor cocktail to BOTH cell suspensions at a 1:100 ratio. 

5. Move immediately to Chapter 6. CRITICAL: Delaying loading of the chip will negatively impact results. 

Chapter 6: Chip Loading 

Materials Required (Pre-prepared):

  • Pre-Thawed IsoCode Chips in Vacuum Sealed Bag from Chapter 4 
  • Stained HeLa Cells at 1 x 106 cells/mL from Chapter 3 (unstimulated) and Chapter 5 (stimulated) 

Methods:

1. Remove IsoCode chips from vacuum sealed bag and place on a flat surface. CRITICAL: Keep protective blue film on bottom of chip. 

2. Resuspend unstimulated cells by gently pipetting up and down 15 times. Immediately proceed to chip loading. Pipette 35 µL of cell suspension into IsoCode chip. CRITICAL: Be careful not to create bubbles. Insert pipette tip vertically into inlet port until tip lightly touches bottom, and slowly pipette 35 µL into inlet port. Be careful not to eject second step of pipette–it will cause bubbles. 

3. Resuspend stimulated cells by gently pipetting up and down 15 times. Immediately proceed to chip loading. Pipette 35 µL of cell suspension into IsoCode chip. CRITICAL: Be careful not to create bubbles. Insert pipette tip vertically into inlet port until tip lightly touches bottom, and slowly pipette 35 µL into inlet port. Be careful not to eject second step of pipette–it will cause bubbles. 

4. Let IsoCode chips sit for one minute on a flat surface. 

5. Check bottom of chip to ensure liquid has entered the chip. TIP: If liquid has not flowed, tap IsoCode chip on flat surface lightly. 

6. When inserting IsoCode chip into IsoLight, make sure barcode is facing up and towards you with the magnet facing the IsoLight. Take the blue film off while inserting each IsoCode chip into the IsoLight. NOTE: Please refer to the loading instructions in IsoLight system guide for detailed instructions. 


Notes and Comments

D. Appendix 

D1 Protocol: Cell Quantification & Viability 

Materials Required:

  • Hemocytometer
  • 10 µL aliquot of cells
  • Trypan Blue

NOTE: Automated cell counters can be used in this protocol EXCEPT prior to loading cells on chip due to spectral overlap of the stains. Manual cell counting is required prior to loading on the chip. 

1. Using a P10 pipette, add equal volume of Trypan blue solution to 10 µL of sample. Mix gently to resuspend. TIP: Make sure to pipette around the tube to ensure there are no clumps or bubbles. 

2. Load onto hemocytometer. CRITICAL: Be careful not to overfill or create bubbles. 

3. Count and record viable (clear) and dead cells (blue) of all four 16-square corners. 

CRITICAL: If more than 200 cells/16 squares were counted, repeat count using a 1:5 or 1:10 dilution with 1X PBS or complete RPMI using a fresh sample aliquot. 

4. Calculate the concentration of cells as follows: 

  • Concentration (cells/mL) = Average per square cell count x 104 x dilution factor

5. Calculate the number of cells as follows: 

  • Number of cells = Cell concentration (cells/mL) from D.1.3 x total volume of cell suspension (mL)

6. Calculate percent viable cells:

  • % Viable cells = 100 x number of viable cells / [number of viable cells + number of dead cells]

For troubleshooting, see page 18 on the PDF of the protocol, or contact Support at 475-221-8402 & support@isoplexis.com with specific troubleshooting questions. 


References

IsoPlexis