Home3D Cell CultureCultrex® 3-D Culture Cell Harvesting Kit Protocol

Cultrex® 3-D Culture Cell Harvesting Kit Protocol

For harvesting and lysate preparation from 3D Culture for Western analysis

Product Number 3448-020-K

I. Product Description

3D Cultures exhibit cellular behaviors and morphologies similar to those seen in vivo, however, the adaptation of these models for studying biochemical processes has been impeded by the challenge of separating intact cells from extra-cellular proteins comprising the hydrogel. Commonly, proteases are employed to degrade these extracellular proteins, however, proteases also degrade proteins on the cell surface and protease activity may carry over into lysate preparations. Non-enzymatic methodologies have also been described for depolymerizing extracellular matrix proteins, although the implementation of these protocols remains problematic for some researchers. Cultrex® 3D Culture Cell Harvesting Kit provides an optimized and standardized solution for the isolation and normalization of cell lysates from 3-D Culture Matrix BME or Laminin I for subsequent biochemical analysis.

II. Components

III. Reagents and Equipment Required but not Provided

  1. Equipment
    1. Low speed swinging bucket 4 ⁰C centrifuge and tubes for cell harvesting
    2. 4 ⁰C storage
    3. Ice bucket
    4. Timer
    5. Orbital Shaker
  2. Reagents
    1. Deionized water
    2. Western Blocking Reagent (WESTBL-RO), Tris Buffered Saline, with TWEEN® 20, pH 8.0 (T9039) or 5% milk
  3. Disposables
    1. Centrifuge tubes, 150 mL (T1943)
    2. Serological pipettes, 1, 5, and 10 mL (SIAL1485, SIAL1487, SIAL1488)
    3. Gloves

IV. Precautions and Limitations

  1. For Research Use Only. Not for use in diagnostic procedures.
  2. The physical, chemical, and toxicological properties of these products may not yet have been fully investigated; therefore, we recommend the use of gloves, lab coats, and eye protection while using these chemical reagents.
  3. The CULTREX® 3D Culture Cell Harvesting Kit contains reagents that may be harmful if swallowed, or come in contact with skin or eyes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.

V. Preparation Instructions

  1. 10X Cell Harvesting Buffer
    Dilute 1:10 in deionized water and chill to 4 ⁰C prior to use.
  2. 10X Cell Wash Buffer
    Dilute 1:10 in deionized water and chill to 4 ⁰C prior to use.
  3. 10X Loading Buffer
    Dilute 1:10 in cell lysates for western blotting.
  4. Rabbit Anti-G3PDH Polyclonal Antibody
    Dilute 1:1000 in blocking buffer, such as TBST, 5% milk, and use immediately. Store unused antibody in aliquots at –20°C. Avoid freeze/thaws.

VI. Assay Protocol

These procedures should be performed in a biological hood utilizing aseptic technique to prevent contamination.

A. Cell Harvesting

Optimal depolymerization of BME or Laminin I requires at least a five-fold excess of 1X Cell Harvesting Buffer and is more efficient with larger ratios. The reaction should be performed in 15 ml conical tubes for optimal cell pelleting. Larger volumes and high density hydrogels may require dividing samples into multiple tubes for optimal depolymerization.

  1. For each 35 mm plate, dilute buffers and chill overnight at 4 ⁰C.
    1. Dilute 5 ml of 10X Wash Buffer to 50 ml with dH2O.
    2. Dilute 5 ml of 10X Cell Harvesting Buffer to 50 ml with dH2O.
    3. Keep buffers on ice throughout procedure.
  2. Working on ice, aspirate cell culture media, and gently wash dish three times with 10 mL of 1X Wash Buffer. Be very careful not to disturb hydrogel; if the hydrogel appears to break up, transfer washes to a 15 ml conical tube, centrifuge off wash solution, and use this tube in step 15.
  3. After final wash, aspirate buffer, and add 6 ml of 1X Cell Harvesting Buffer to dish.
  4. Resuspend cells and matrix in Cell Harvesting Buffer by gently pipetting up and down using a serological pipet.
  5. Once resuspended, transfer contents to a 15 mL conical tube.
  6. Transfer cells to a 15 ml conical tube, and add 5 ml of cell culture medium.
  7. Seal the tube and place on its side in an ice container.
  8. Gently shake container for 30 minutes on an orbital shaker.
  9. Centrifuge conical tube at 200 x g for 5 minutes at 4 ⁰C, and aspirate supernatant. If a thick layer of hydrogel is still visible, then wash with additional 10 ml 1X Cell Harvesting Buffer, centrifuge at 200 x g for 5 minutes at 4 ⁰C, and aspirate supernantant; repeat if necessary. It is important to keep cell samples and Cell Harvesting Buffer on ice during processing.
  10. Reagents are supplied for preparation and normalization of lysates for western blot; alternatively, cells may be processed for RNA or DNA isolation using TRI Reagent® (T9424).
Morphology of MCF-10A (A,B) and MCF-7 cells (C,D)

Figure 1. Morphology of MCF-10A (A,B) and MCF-7 cells (C,D) in traditional 2D Culture and 3D BME Culture, Scale = 250 µm

Harvesting Cells From 3D Culture.

Figure 2.Harvesting Cells From 3D Culture.

B. Lysate Preparation

  1. Re-suspend cell pellet in 50 ml of Sample Buffer, and pipette up and down to aid in cell lysis. Additional Sample Buffer may be necessary to solubilize large cell pellets. Protease inhibitors may also be added at this time, if needed.
  2. Incubate lysates at 95 ⁰C for 5 minutes.
  3. Assay lysates for protein concentration. OD280 and BCA assays have resulted in reproducible results.
  4. Determine dilution for lysate mass:
    Lysate concentration (µg/µl) X Lysate volume (µl) = Lysate mass (µg)

  5. Determine final volume:
    Lysate mass (µg) / Desired concentration (µg/µL) = Final volume

  6. Determine volume of 10X loading buffer needed:
    Final volume (µL) X 0.1=10X Loading buffer (µL)

  7. Determine volume of sample buffer needed:
    [Final volume (µL) – Lysate volume (µL)] – 10X Loading Buffer (µL) = Sample buffer (µL)

  8. Normalize lysate loading by evaluating sample via Western Blot using a 1:1000 dilution of rabbit anti-G3PDH polyclonal antibody. Antibody must be used immediately after dilution; unused antibody should be stored in aliquots at –20°C.
  9. Use normalized volumes for assessing targets of choice.

C. Sample Data

 Evaluation of RNA expression via RT-PCR (A) and protein expression

Figure 3. Evaluation of RNA expression via RT-PCR (A) and protein expression via Western blotting (B) for GAPDH from MCF-10A and MCF-7 mammary epethelial cells isolated from either traditional 2D culture or 3D BME culture.