THP-1 Cell Culture: A Comprehensive SOP and Key Considerations

THP-1 Cell Culture: A Comprehensive SOP and Key Considerations

THP-1 is a human monocytic leukemia cell line widely used as a suspension model for monocyte–macrophage differentiation, inflammation, and immunology research. The cells grow in suspension; in a healthy state they are uniform in size with strong refractility, and a fraction naturally aggregate into small clusters. THP-1 cells are highly sensitive to serum, medium, and cell density, and their status is easily affected by the environment, handling, and serum quality. This article outlines and guides the THP-1 cell culture workflow.

I. Cell Thawing

1. Preparation: Pre-warm the water bath to 37°C; prepare 9 mL of medium and pre-warm it to 37°C; irradiate the biosafety cabinet with UV for 30 min in advance.
2. Thawing: Remove the cryovial from liquid nitrogen and rapidly agitate it in a 37°C water bath until melted (~1–2 min).
3. Resuspension: Wipe the cryovial exterior with 75% ethanol, open it inside the biosafety cabinet, and slowly drip the cell suspension into a 15 mL centrifuge tube containing 9 mL of pre-warmed complete medium.
4. Centrifugation: Centrifuge at 600 rpm for 5 min and discard the supernatant.
5. Resuspension: Add 1 mL of complete medium and gently pipette up and down ~10 times to resuspend the cells. Prepare a new 10 cm dish, labeling the lid with cell name, passage operator, passage number, and date. Add 9 mL of complete medium, then add 1 mL of the cell suspension. Mix by drawing a cross pattern to distribute cells evenly.
6. Culture: Place the dish into the incubator for culture.

II. Cell Passaging

1. Preparation: Prepare complete medium: RPMI-1640 basal medium (abs9484-500mL) + 10% FBS (abs972-500mL) + 1% penicillin–streptomycin (P/S) (abs9244-100mL); take out PBS (abs962-500mL) and complete medium in advance and bring to room temperature; irradiate the biosafety cabinet with UV for 30 min.

   Note: All reagents used during passaging must be brought to room temperature.

2. Microscopic inspection: Observe the cells under a microscope; passage when the cell density reaches ~80%, large numbers of suspended cells are released upon gentle dish agitation, and the medium color has slightly yellowed.
3. Cell collection: Collect all cell suspension into a 15 mL centrifuge tube.
4. Centrifugation: Centrifuge at 600 rpm for 5 min, aspirate and discard the supernatant with a pipette tip; add 3 mL of complete medium and gently pipette ~10 times to resuspend the cells.

   Note: If cell status is poor or cellular debris is abundant, after discarding the supernatant at this step add 10 mL of PBS, pipette to resuspend, centrifuge at 600 rpm to discard the supernatant, then proceed with passaging.

   Note: This cell line is sensitive to handling; avoid vigorous pipetting, which can damage cells.

   Note: Recommended split ratio is 1:3–1:4; add an appropriate volume of complete medium according to the split ratio.

5. Passaging: Prepare a new 10 cm dish, labeling the lid with cell name, passage operator, passage number, and date. Add 9 mL of complete medium, then add 1 mL of the cell suspension. Mix by drawing a cross pattern to distribute cells evenly.
6. Microscopic inspection: Observe cell status and even distribution under the microscope, then place in a 37°C, 5% CO₂ incubator; inspect cell status every 24 h.

   Note: The typical passaging density is 1:3. Low density slows proliferation and degrades cell status. This cell line is sensitive to serum; premium-grade Absin FBS is recommended, as poor-quality serum easily causes cells to develop elongated, "horned" morphologies.

III. Cell Cryopreservation

1. Preparation: Use a serum-free cell freezing medium (abs9417-100mL), or prepare your own: 90% FBS (abs972-500mL) + 10% DMSO (abs9187-100mL). Pre-label the cryovials with cell name, passage number, operator, and date. Irradiate the biosafety cabinet with UV for 30 min.

   Note: A single dish can be frozen into 3 vials.

2. Cell collection: When cell density reaches ~80%, collect cells following the passaging steps; after 600 rpm centrifugation discard the supernatant.
3. Cell resuspension: Add 3 mL of cryopreservation medium and gently pipette to resuspend the cells.
4. Freezing: Add 1 mL of the mixture to each cryovial, place the vials in a freezing container, store in a −80°C freezer overnight, then transfer to a liquid-nitrogen tank.

IV. Key Considerations

1. Strictly control passaging density: The typical split ratio is 1:3. Low density slows proliferation and degrades cell status.
2. Serum and medium selection: Culture with premium-grade serum; poor-quality serum easily causes cells to develop a "horned" morphology.
3. Contamination control: Prone to mycoplasma infection, manifesting as slowed growth and altered morphology; perform regular mycoplasma testing and maintain strict aseptic technique throughout.
4. Handling: This cell line is sensitive to handling; avoid vigorous pipetting, which can damage cells. Bring all reagents to room temperature before passaging.

V. Absin Fetal Bovine Serum

Product Advantages

1. A serum brand under Univ (Youningwei), with literature support for nearly 300 papers (highest IF = 32.4);
2. Stably used by academician-led research groups;
3. Validation data across nearly 20 cell types, performance benchmarked against imported premium brands;
4. Low endotoxin (<3 EU/mL) and low hemoglobin (<50 mg/L);
5. Ample single-batch production capacity with minimal inter-batch variation;
6. Professional technical support and expert cell-culture SOP guidance;
7. 50 mL small-pack aliquot service;
8. Diverse serum types to power your experiments.

Cat. No.	Product Name	Specification	Origin
abs981-500mL	Fetal Bovine Serum (Premium Grade)	500 mL	Inner Mongolia, China
abs972-500mL	Fetal Bovine Serum (Superior Grade)	500 mL	Inner Mongolia, China
abs974-500mL	Fetal Bovine Serum (Standard Grade)	500 mL	Inner Mongolia, China

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