Feb 24, 2026
Cell Culture Procedure
- Herbi Yuliantoro1
- 1Lehigh University
- Herbi
Protocol Citation: Herbi Yuliantoro 2026. Cell Culture Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9bow1l3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 24, 2026
Last Modified: February 24, 2026
Protocol Integer ID: 243970
Keywords: cell culture procedure proteomic, direct infusion
Abstract
Proteomics workflow for Direct infusion
Guidelines
8. Put the new flasks back to grow**
- Place the flasks in the incubator at 37°C with 5% CO2**.
9. Change the medium regularly**
- Replace the medium every 2–3 days**.
10. When to split again**
- Split again when the cells reach about 6–7 × 10^4^ cells per cm² (often around once per week**, depending on growth).
**Proteomics workflow (direct infusion)**
**Step 1: Harvest and Wash Cells**
1. Collect ~5 million cells (~100–300 µg protein yield).
2. Transfer to a 1.5 mL microcentrifuge tube.
3. Spin at 500 × g for 5 min at room temperature.
4. Aspirate medium completely.
5. Wash once with 1.0 mL cold PBS, pipette to mix.
6. Spin again, remove supernatant.
**Step 2: Cell Lysis in 8 M Urea**
1. Add 200 µL of 8 M urea in 100 mM ABC to the washed pellet.
2. Vortex or pipette up/down to lyse.
3. Short sonication (3 × 10 s bursts), see the clarity of the sample; if still sticky, try to sonicate again.
4. Spin at 10,000 × g for 5 min at 4 degrees Celsius.
5. Transfer clear lysate (~190 µL) to a new 1.5 mL tube.
6. Check with Bradford assay.**
**Bradford assay workflow:**
- Turn on Genesys 20 spectrophotometer.
- Use the arrow keys to set the wavelength to 595 nm.
**Prepare blank:**
- In a cuvette, mix: 1.00 mL Bradford reagent +5 µL ddH2O (acts as a zero-protein control)
- Insert into the spectrophotometer and press "0 ABS" to blank it.
**Prepare the sample cuvette:**
- Mix in a new cuvette: o 1.00 mL Bradford reagent o 5 µL of cell lysate
- Mix gently (vortex briefly or pipette up/down).
- Incubate for 5–10 min at room temperature (no light needed). Measure sample:
- Insert the sample cuvette.
- Read the absorbance at 595 nm.
- Record the value (A595).
**Step 3: Reduction and Alkylation**
_Assume 190 µL protein solution:_
3.1 Reduce**
- Add 10 µL of 200 mM DTT.
- Incubate at 37 °C for 30 min.
3.2 Alkylate**
- Add 20 µL of 200 mM IAA.
- Incubate at room temp, dark, 30 min.
3.3 Quench**
- Add another 10 µL of DTT.
- Total volume: ~230 µL
**Step 4: Dilute Urea and Digest with Trypsin**
1. Add 600 µL 100 mM ammonium bicarbonate to reduce urea to 3c2 M or add 700 if the concentration of ABC is 50 mM.
- New volume: ~830 µL
2. Add 30-60 µL of 0.1 µg/µL stock, depending on the concentration from the Bradford assay test. Use 1:50 for complete digestion.
3. Mix gently, cap the tube.
_Incubate overnight (~16 h) at 37 °C in incubator_
**Washing step using the C18 Spin Column**
**Step 1: Preparation of Solutions**
1. Prepare 0.5% Formic Acid in 5% ACN (Equilibration/Wash Solution)**
- Components:**
- 99% Formic Acid: 5 µL
- ACN: 50 µL
- Ultrapure Water: 945 µL
- Procedure:**
1. Measure 5 µL of 99% formic acid using a micropipette.
2. Add it to a container.
3. Add 50 µL of ACN.
4. Add 945 µL of ultrapure water.
5. Mix well to ensure homogeneity.
2. Prepare 2% Formic Acid in 20% ACN (Sample Buffer)**
- Components:**
- 99% Formic Acid: 20 µL
- ACN: 200 µL
- Ultrapure Water: 780 µL
- Procedure:**
1. Measure 20 µL of 99% formic acid and add it to a container.
2. Add 200 µL of ACN.
3. Add 780 µL of ultrapure water.
4. Mix well.
3. Prepare 70% ACN with 0.1% Formic Acid (Elution Buffer)**
- Components:**
- ACN: 700 µL
- 99% Formic Acid: 1 µL
- Ultrapure Water: 299 µL
- Procedure:**
1. Measure 700 µL of ACN and add it to a container.
2. Add 1 µL of 99% formic acid.
3. Add 299 µL of ultrapure water.
4. Mix thoroughly.
**Step 2: Eluting the Sample Using Pierce™ C18 Spin Column**
1. Activation:**
- Procedure:**
1. Add 200 µL of 50% methanol (or ACN) to the spin column.
2. Place the column into a microcentrifuge tube.
3. Centrifuge at 1,500 x g (423 RPM) for 1 minute.
4. Discard the flow-through.
5. Repeat the activation step once more.
2. Equilibration:**
- Procedure:**
1. Add 200 µL of 0.5% formic acid in 5% ACN to the column.
2. Centrifuge at 1,500 x g for 1 minute.
3. Discard the flow-through.
4. Repeat the equilibration step once more.
3. Sample Loading:**
- Procedure:**
1. Prepare the Sample:**
- Mix 200 µL of your peptide sample with 66.7 µL of 2% formic acid in 20% ACN.
- This yields a total volume of 266.7 µL with a final concentration of 0.5% formic acid in 5% ACN.
2. Load the Sample:**
- Add the 266.7 µL sample onto the top of the resin bed in the spin column.
- Centrifuge at 1,500 x g for 1 minute.
- Collect the flow-through.
3. Reload the Sample:**
- To ensure complete binding, reload the flow-through onto the column and centrifuge again.
- Repeat this process 2-3 times.
4. Discard or Retain Flow-Through:**
- After ensuring binding, discard the flow-through or retain a small portion for verification.
4. Washing:**
- Procedure:**
1. Add 200 µL of 0.5% formic acid in 5% ACN to the column.
2. Centrifuge at 1,500 x g for 1 minute.
3. Discard the flow-through.
4. Repeat the wash step once more.
5. Elution:**
- Procedure:**
1. Add 20 µL of 70% ACN with 0.1% formic acid to the column.
2. Centrifuge at 1,500 x g for 1 minute.
3. Collect the eluate in a clean microcentrifuge tube.
4. Repeat the elution step with another 20 µL to ensure complete elution.
5. Combine the eluates if necessary.
6. Lyophilization**
- Put your microcentrifuge in the vacuum concentrator (SpeedVac) or Freeze Dryer container to dry it overnight.
Materials
- D-PBS (Dulbecco’s Phosphate-Buffered Saline)
- Trypsin-EDTA
- Complete medium (EMEM + 10% FBS)
- EMEM
- FBS
- Fresh complete medium
- Incubator with 5% CO2
- HEK293: EMEM + 10% FBS
- HEK293T: DMEM + 10% FBS
- MCF-7: EMEM + 10% FBS + insulin
- 8 M Urea
- 100 mM ABC
- Bradford reagent
- ddH2O
- Cuvette
- Genesys 20 spectrophotometer
Troubleshooting
Cell Culture Procedure
Throw away the old liquid
- Carefully remove the old nutrient liquid from the flask.
Quick rinse
- Add a small amount of D-PBS (Dulbecco’s Phosphate-Buffered Saline).
- Gently swirl once so it touches the cell layer.
- Pour it out.
Add the liquid that helps cells let go
- Add 2–3 mL Trypsin-EDTA.
- Make sure it covers the bottom surface where the cells are.
Wait until the cells release
- Look under the microscope and wait until the cells lift off (usually 5–15 minutes).
- Do not hit or shake the flask (this can cause clumps).
- If cells are slow to lift, keeping the flask at 37°C can help.
Stop the Trypsin-EDTA
- Add 6–8 mL complete medium to stop the Trypsin-EDTA.
- Complete medium = EMEM + 10% FBS
- EMEM = base nutrient liquid
- FBS = serum (helps cells grow)
Mix the cells evenly
- Gently pipette up and down to make the cell liquid evenly mixed (avoid bubbles).
Move cells into new flasks/plates
1. total mixed cell liquid is about 10 mL:
- putting 2 mL into a new flask means you moved about 1/5 of the cells, for a more diluted split like 1:6 to 1:10, you would use less than 2 mL from that same 10 mL total.
2. After adding the cell liquid to the new flask, add 8 mL fresh complete medium to reach your normal final volume around 10mL.
Put the new flasks back to grow
- Place the flasks in the incubator at 37°C with 5% CO₂.
Change the medium regularly
- Replace the medium every 2–3 days.
When to split again
- Split again when the cells reach about 6–7 × 10^4^ cells per cm² (often around once per week, depending on growth).
Note:
- HEK293**: EMEM + 10% FBS
- HEK293T**: DMEM + 10% FBS
- MCF-7**: EMEM + 10% FBS + insulin
Proteomics workflow (direct infusion)
Step 1: Harvest and Wash Cells
Collect ~5 million cells (~100–300 µg protein yield).
Transfer to a 1.5 mL microcentrifuge tube.
Spin at 500 × g for 5 min at room temperature.
Aspirate medium completely.
Wash once with 1.0 mL cold PBS, pipette to mix.
Spin again, remove supernatant.
Step 2: Cell Lysis in 8 M Urea
Add 200 µL of 8 M urea in 100 mM ABC to the washed pellet.
Vortex or pipette up/down to lyse.
Short sonication (3 × 10 s bursts), see the clarity of the sample; if still sticky, try to sonicate again.
Spin at 10,000 × g for 5 min at 4 degrees Celsius.
Transfer clear lysate (~190 µL) to a new 1.5 mL tube.
Check with Bradford assay.
Bradford assay workflow: Turn on Genesys 20 spectrophotometer.
Use the arrow keys to set the wavelength to 595 nm.
Prepare blank:
- In a cuvette, mix: 1.00 mL Bradford reagent + 5 µL ddH₂O (acts as a zero-protein control)
- Insert into the spectrophotometer and press "0 ABS" to blank it.
Prepare the sample cuvette:
- Mix in a new cuvette: 1.00 mL Bradford reagent + 5 µL of cell lysate
- Mix gently (vortex briefly or pipette up/down).
- Incubate for 5–10 min at room temperature (no light needed).
Measure sample:
- Insert the sample cuvette.
- Read the absorbance at 595 nm.
- Record the value (A595).
Step 3: Reduction and Alkylation
Assume 190 µL protein solution:
3.1 Reduce
- Add 10 µL of 200 mM DTT.
- Incubate at 37 °C for 30 min.
3.2 Alkylate
- Add 20 µL of 200 mM IAA.
- Incubate at room temp, dark, 30 min.
3.3 Quench
- Add another 10 µL of DTT.
- Total volume: ~230 µL
Step 4: Dilute Urea and Digest with Trypsin
Add 600 µL 100 mM ammonium bicarbonate to reduce urea to 3c2 M or add 700 if the concentration of ABC is 50 mM.
- New volume: ~830 µL
Add 30-60 µL of 0.1 µg/µL stock, depending on the concentration from the Bradford assay test. Use 1:50 for complete digestion.
Mix gently, cap the tube.
Incubate overnight (~16 h) at 37 °C in incubator
Washing step using the C18 Spin Column
Step 1: Preparation of Solutions
Prepare 0.5% Formic Acid in 5% ACN (Equilibration/Wash Solution)
Components:
- 99% Formic Acid: 5 µL
- ACN: 50 µL
- Ultrapure Water: 945 µL
Procedure:
1. Measure 5 µL of 99% formic acid using a micropipette.
2. Add it to a container.
3. Add 50 µL of ACN.
4. Add 945 µL of ultrapure water.
5. Mix well to ensure homogeneity.
Prepare 2% Formic Acid in 20% ACN (Sample Buffer)
Components:
- 99% Formic Acid: 20 µL
- ACN: 200 µL
- Ultrapure Water: 780 µL
Procedure:
1. Measure 20 µL of 99% formic acid and add it to a container.
2. Add 200 µL of ACN.
3. Add 780 µL of ultrapure water.
4. Mix well.
Prepare 70% ACN with 0.1% Formic Acid (Elution Buffer)
Components:
- ACN: 700 µL
- 99% Formic Acid: 1 µL
- Ultrapure Water: 299 µL
Procedure:
1. Measure 700 µL of ACN and add it to a container.
2. Add 1 µL of 99% formic acid.
3. Add 299 µL of ultrapure water.
4. Mix thoroughly.
Step 2: Eluting the Sample Using Pierce™ C18 Spin Column
Activation:
Procedure:
1. Add 200 µL of 50% methanol (or ACN) to the spin column.
2. Place the column into a microcentrifuge tube.
3. Centrifuge at 1,500 x g (423 RPM) for 1 minute.
4. Discard the flow-through.
5. Repeat the activation step once more.
Equilibration:
Procedure:
1. Add 200 µL of 0.5% formic acid in 5% ACN to the column.
2. Centrifuge at 1,500 x g for 1 minute.
3. Discard the flow-through.
4. Repeat the equilibration step once more.
Sample Loading:
Procedure:
1. Prepare the Sample:
- Mix 200 µL of your peptide sample with 66.7 µL of 2% formic acid in 20% ACN.
- This yields a total volume of 266.7 µL with a final concentration of 0.5% formic acid in 5% ACN.
Load the Sample:
- Add the 266.7 µL sample onto the top of the resin bed in the spin column.
- Centrifuge at 1,500 x g for 1 minute.
- Collect the flow-through.
Reload the Sample:
- To ensure complete binding, reload the flow-through onto the column and centrifuge again.
- Repeat this process 2-3 times.
Discard or Retain Flow-Through:
- After ensuring binding, discard the flow-through or retain a small portion for verification.
Washing:
Procedure:
1. Add 200 µL of 0.5% formic acid in 5% ACN to the column.
2. Centrifuge at 1,500 x g for 1 minute.
3. Discard the flow-through.
4. Repeat the wash step once more.
Elution:
Procedure:
1. Add 20 µL of 70% ACN with 0.1% formic acid to the column.
2. Centrifuge at 1,500 x g for 1 minute.
3. Collect the eluate in a clean microcentrifuge tube.
4. Repeat the elution step with another 20 µL to ensure complete elution.
5. Combine the eluates if necessary.
Lyophilization
- Put your microcentrifuge in the vacuum concentrator (SpeedVac) or Freeze Dryer container to dry it overnight.