Preparation of Subcellular Fractions from Leukemic Cells: A Method to Generate Cellular and Nuclear Fractions from Leukemic Cells

1. Preparation of Subcellular Fractions from CLL Cells

NOTE: When planning the experimental set-up, include a well of unstimulated/untreated cells from which the whole cell extract can be generated.

1. Perform the desired stimulation and/or drug treatment of the MEC1 CLL cell line or isolated primary CLL cells using 10 – 20 x 10⁶ cells/condition. Cells will then be used for subcellular fractionation or to generate whole cell extract as explained below:
2. Preparation of solutions/tubes: Prepare all solutions/buffers freshly on the day of the fractionation before the cells are harvested. Store the solutions on ice until required and use within 4 h of preparation.
   1. PBS/phosphatase inhibitor solution: Prepare the phosphatase inhibitors in PBS by diluting the phosphatase inhibitors 1:20 in 1x PBS (i.e., 0.5 mL of phosphatase inhibitors in 9.5 mL of 1x PBS).
      NOTE: Ensure the phosphatase inhibitors have not precipitated. If a precipitate is present, heat to 50 °C for 10 min.
   2. Hypotonic buffer: Prepare 1x hypotonic Buffer by making a 1:10 dilution of 10x hypotonic buffer in distilled water (i.e., 50 μL of 10x hypotonic Buffer into 450 μ L of dH₂O).
   3. 10 mM dithiothreitol (DTT): Prepare 10 mM DTT by making a 1:100 dilution of 1M DTT with distilled water (i.e., 10 μL of 1 M DTT in 990 μL of dH₂O).
      NOTE: DTT is highly labile so prepare this freshly each time. Avoid repeated freeze/thaw cycles.
   4. Complete lysis buffer: Determine how much buffer is required for each experiment. Each sample requires 50 μL of complete lysis buffer, so add 5 μL of 10 mM DTT to 44.5 μL of lysis buffer and then add 0.5 μL of protease inhibitor cocktail. This amount can be scaled up depending on the number of samples in the experiment.
   5. Label four sets of 1.5 mL microfuge tubes for each stimulation and/or drug treatment for the freshly stimulated cells, the freshly generated cytoplasmic fractions, the freshly generated nuclear fractions, and the whole cell lysates. Pre-chill these microfuge tubes on ice until required.
3. Transfer the cells into individually labeled 1.5 mL microfuge tubes and pellet by centrifuging at 200 x g for 5 min at 4 °C. Remove the supernatant and resuspend the cells in 1 mL of ice-cold PBS/phosphatase inhibitors. Pellet the cells by centrifugation at 200 x g for 5 min at 4 °C. Remove the supernatant and keep the cell pellets on ice.
4. Preparation of cytoplasmic fractions: Gently resuspend the cell pellets to be used for subcellular fractionation in 50 μL of 1x hypotonic Buffer. Incubate the cells on ice for 15 min to allow the cells to swell.
   NOTE: The volume of hypotonic buffer used can be increased empirically depending on the cell number.
   1. Add 0.8 – 2.5 μL (1:20 to 1:60) of detergent into each sample and vortex on the highest setting for 10 s.
      1. To determine the optimal concentration of detergent to use for a specific cell type to isolate nuclear and cytoplasmic fractions, perform a detergent gradient initially. A range of 1:20 to 1:60 (i.e., 2.5 μL to 0.8 μL of detergent into 50 μL of Hypotonic Buffer) should be adequate.
         NOTE:  If the volume of hypotonic buffer in step 3.4 is adjusted, ensure the appropriate detergent ratio is maintained.
      2. Verify cell lysis by observing cells using a phase contrast microscope before and after addition of detergent. Whole cells appear larger with a dense, dark nucleus. The cytoplasm will appear as a bright halo around the nucleus.
         NOTE: Appropriate lysis is further confirmed by using Western blotting to analyze specific proteins within the lysed fractions generated from the detergent gradient.
   2. Once lysed, centrifuge the samples at 14,000 x g for 30 s at 4 °C.
   3. Carefully transfer the supernatant into a pre-chilled, labeled microfuge tube. This cytoplasmic fraction can be stored at -80 °C until required for further analysis. The remaining pellet contains the nuclear fraction.
      NOTE: Avoid repeated freeze/thaw cycles of the samples.
5. Preparation of nuclear fractions: Resuspend each nuclear pellet in 50 μL of complete lysis buffer by pipetting up and down.
   NOTE: The volume of complete lysis buffer can be adjusted empirically according to the starting cell number.
   1. Add 2.5 μL of detergent to solubilize proteins associated with the nuclear membrane and vortex on the highest setting for 10 s. Incubate the samples on ice for 30 min.
   2. Vortex on the highest setting for 30 s, then centrifuge the samples at 14,000 x g for 20 min at 4 °C.
   3. Transfer the supernatant into a pre-chilled, labeled microfuge tube. This nuclear fraction can be stored at -80 °C until required for further analysis.
      NOTE: Avoid repeated freeze/thaw cycles of the samples.