Jurkat Cell Culture: A Step-by-Step SOP and Notes

Jurkat is a human T-lymphoblastic leukemia cell line, a widely used suspension cell model for investigating T-cell activation, inflammation and immune signaling pathways. Jurkat cells tend to form natural cell aggregates and are highly sensitive to serum, cell density and mechanical pipetting. Their viability is easily affected by the culture environment, manual operations and serum quality. This document summarizes the standard operating procedures and guidance for Jurkat cell culture.

1. Cell Thawing

1. Preparation: Preheat a water bath to 37°C; aliquot 9 mL of complete medium and warm it to 37°C; turn on UV irradiation for the biosafety cabinet for 30 min in advance.
2. Thawing: Retrieve the cryovial from liquid nitrogen and rapidly thaw the contents with gentle agitation in a 37°C water bath for approximately 1–2 min.
3. Resuspension: Wipe the exterior of the cryovial with 75% ethanol. Open the vial inside the biosafety cabinet, and slowly add the cell suspension dropwise into a 15 mL centrifuge tube preloaded with 9 mL pre-warmed complete medium.
4. Centrifugation: Centrifuge at 600 rpm for 5 min and discard the supernatant.
5. Resuspension: Add 1 mL complete medium and gently pipette up and down 10 times to resuspend the cell pellet. Prepare a new 10 cm culture dish and label the lid with cell line name, operator, passage number and date. Add 9 mL complete medium followed by 1 mL cell suspension. Mix by cross-rocking to achieve uniform cell distribution across the dish.
6. Incubation: Place the dish into the cell incubator.

2. Cell Passaging

1. Experimental 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 equilibrate to room temperature. Perform UV disinfection of the biosafety cabinet for 30 min beforehand.
2. Microscopic Examination: Observe cells under a microscope. Passage is recommended when cell density reaches ~80%, abundant suspended cells are observed upon gentle shaking of the dish, and the culture medium turns slightly yellow.
3. Cell Harvesting: Transfer all cell suspension into a 15 mL centrifuge tube.
4. Centrifugation: Centrifuge at 600 rpm for 5 min and aspirate the supernatant. Add 3 mL complete medium and gently pipette up and down around 10 times to resuspend cells.

   Note: If cells are in poor condition or abundant cell debris exists, add 10 mL PBS for resuspension after supernatant removal, centrifuge at 600 rpm and discard supernatant before passaging.

   Note: This cell line is sensitive to handling. Avoid vigorous pipetting, as it may cause cell damage.

   Note: The recommended split ratio is 1:3 to 1:4. Add an appropriate volume of complete medium according to the split ratio.

5. Subculture: Prepare a new 10 cm culture dish and label the lid with cell line name, operator, passage number and date. Add 9 mL complete medium followed by 1 mL cell suspension. Mix by cross-rocking to distribute cells evenly.
6. Microscopic Check: Examine cell morphology and uniformity under a microscope. Place the dish in a 37 °C, 5% CO₂ incubator. Monitor cell status every 24 h.

   Note: Jurkat cells are serum-sensitive. absin premium FBS is recommended; low-quality serum leads to slow cell proliferation.

3. Cell Cryopreservation

1. Preparation: Serum-free cryopreservation medium (abs9417-100mL) can be used. Alternatively, prepare homemade freezing medium: 90% FBS (abs972-500mL) + 10% DMSO (abs9187-100mL). Pre-label cryovials with cell line name, passage number, operator and freezing date. Sterilize the biosafety cabinet with UV light for 30 min in advance.

   Note: Jurkat cells are sensitive to freezing density. It is recommended to prepare 2–3 cryovials per 10 cm dish.

2. Cell Harvesting: Harvest cells at ~80% confluency following the passaging protocol, then centrifuge at 600 rpm and discard supernatant.
3. Cell Resuspension: Add 3 mL cryopreservation medium and gently pipette to resuspend cells.
4. Cryostorage: Aliquot 1 mL cell mixture into each cryovial. Place cryovials into a freezing container and store at -80 °C overnight. Transfer vials to liquid nitrogen tank the next day for long-term storage.

4. Critical Precautions

• Serum Selection
  Jurkat cells are serum-sensitive. Premium-grade serum is required. Inferior serum results in retarded cell growth.
• Contamination Control
  Jurkat is susceptible to mycoplasma infection, manifested as slow proliferation and deteriorated cell morphology. Conduct routine mycoplasma testing and maintain strict aseptic technique throughout all operations.
• Operation Notes
  This cell line is sensitive to manipulation. Never pipette vigorously, which will induce cell injury.

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