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Cell Culture Protocol

Cell culture entails cultivating and sustaining cells outside their native environment. The reliability of your experimental results hinges on creating optimal conditions for cell growth, which requires careful consideration of various factors, including cell source, cell type, and culture conditions. It's important to note that culture conditions differ for each cell type. Deviations in the culture conditions required for specific cell types can lead to abnormal phenotypes or even complete cell culture failure. Therefore, we recommend you familiarize yourself with the cell line of interest and strictly follow the instructions for each product used in your experiment.

Tab.1 The essential components in cell culture.

Materials Needed Description
Culture Medium Appropriate medium for your cell line, e.g., DMEM, RPMI-1640 with supplements (e.g., FBS, antibiotics).
Trypsin-EDTA Solution For cell detachment.
PBS Sterile, without calcium and magnesium.

Adherent Cell Culture

  1. Prepare Medium
    Mix the basal medium (e.g., DMEM) with 10% FBS and 1% Penicillin-Streptomycin.
  2. Thawing Cells
    Quickly thaw the frozen cell vial in a 37°C water bath.
    Transfer the cell suspension into a 15 mL conical tube containing 10 mL pre-warmed medium.
    Centrifuge the tube at 1000 rpm for 5 minutes to pellet the cells.
    Discard the supernatant and resuspend the cell pellet in a fresh medium before transferring it to a culture flask.
  3. Culturing Cells
    Incubation: Place the culture flask in a 37°C, 5% CO₂ incubator.
    Observation: Use a microscope to observe cell growth and morphology regularly.
  4. Subculturing Cells
    Remove Medium: Aspirate the old medium from the flask.
    Wash Cells: Wash cells with PBS to remove residual medium.
    Trypsinization: First, remove the spent medium by aspiration and rinse the cells with PBS to eliminate residual serum. Next, add enough trypsin-EDTA solution to cover the cell monolayer fully, and then incubate at 37°C for 2-5 minutes, tapping the flask gently to help detach the cells.
    Neutralize Trypsin: Add fresh medium to neutralize trypsin and gently pipette to resuspend cells.
    Centrifuge and Resuspend: Centrifuge the cell suspension at 1000 rpm for 5 minutes, remove the supernatant and resuspend in a fresh medium.
  5. Split Cells
    Transfer the resuspended cells to new flasks at the appropriate ratio (e.g., 1:3 or 1:5).

Suspension Cell Culture

  1. Medium Preparation
    Mix the basal medium (e.g., RPMI 1640) with 10% FBS and 1% Penicillin-Streptomycin.
  2. Thawing Cells
    Thaw Cells: Quickly thaw the frozen cell vial in a 37°C water bath.
    Transfer the cell suspension to a 15 mL conical tube containing 10 mL of pre-warmed medium.
    Centrifuge the tube at 1000 rpm for 5 minutes to pellet the cells.
    Carefully remove the supernatant and resuspend the cell pellet in a fresh medium. Then, transfer the resuspended cells to a culture flask.
  3. Culturing Cells
    Incubation: Place the culture flask in a 37°C, 5% CO₂ incubator.
    Observation: Use a microscope to observe cell growth and morphology regularly.
  4. Subculturing Cells
    Collect Cells: Transfer the cell suspension to a conical tube and centrifuge at 1000 rpm for 5 minutes.
    Remove Supernatant:
    Aspirate the supernatant.
    Resuspend Cells: Resuspend the cell pellet in a fresh medium.
  5. Split Cells
    Transfer the resuspended cells to new flasks at the appropriate ratio (e.g., 1:2 or 1:3).

Notes:

  1. Ensure all solutions and media are pre-warmed to 37℃ before use.
  2. Work quickly but carefully to maintain sterility and cell viability.
  3. Monitor cells regularly under the microscope for contamination and proper growth.
  4. Always label flasks and vials with essential information, such as the cell line, passage number, and date.

SERVICES

Creative Biolabs can customize the appropriate cell culture service according to your experimental needs.

Disclaimer

This procedure should be used only as a guide. Please note that Creative Biolabs does not guarantee the client's cell culture status.


All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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