Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

The use of primary samples from CLL patients described here have been approved by the West of Scotland Research Ethics Service, NHS Greater Glasgow and Clyde (UK) and all work was carried out in accordance with the approved guidelines.

1. Isolation of CLL Cells from Patient Blood Samples

1. Peripheral blood samples from previously consented CLL patients are received from the clinic in EDTA blood collection tubes, accompanied by the white cell count (WCC). Purify the peripheral blood CLL samples according to the WCC. For WCC < 40 x 10⁶ cells/mL, proceed to step 1.1.1; for WCC ≥ 40 x 10⁶ cells/mL, proceed to step 1.1.2.
   1. Pour the contents of all the EDTA blood tubes into a 50 mL conical centrifuge tube and add 50 µL of Human B Cell Enrichment Cocktail per 1 mL of blood. Incubate at room temperature (RT) for 20 min. Proceed to step 1.1.2.
   2. Dilute the sample at a ratio of 1:1 with RT CLL wash buffer (phosphate-buffered salin (PBS), 0.5% Fetal Bovine Serum (FBS) and 2 mM EDTA).
2. Aliquot RT density gradient media into an appropriately sized conical centrifuge tube for the sample (10 mL into a 50 mL tube for 30 mL of sample or 4 mL into a 15 mL tube for 10 mL of sample).
3. Carefully layer the sample on top of the density gradient media and centrifuge at 400 x g for 30 min at RT.
   NOTE: Ensure that the centrifuge is at RT before the samples are placed in the centrifuge as a change in temperature will result in poor enrichment of mononuclear cells, and switch off the brake on the centrifuge, as sudden braking can disrupt the liquid interface.
4. Gently harvest the white layer of mononuclear cells that collect at the interface of the density gradient media and CLL wash buffer, into a fresh 50 mL conical centrifuge tube using a plastic Pasteur pipette.
5. Add 40 mL of CLL wash buffer to the isolated monolayer to wash the cells and centrifuge at 300 x g for 10 min at RT.
6. Discard the supernatant, resuspend the pellet by flicking the bottom of the tube, then repeat the wash step described in step 1.5.
7. Discard the supernatant, resuspend the pellet as described in step 1.6, then resuspend the pellet in a set volume of CLL wash buffer (up to 40 mL, depending on the size of the cell pellet).
8. Count the cells using trypan blue and a haemocytometer. Then proceed to flow cytometry to check the purity of CLL cells.
   NOTE: At this stage the CLL cells can be cultured at a concentration of 10 x 10⁶ cells/mL in media to be used in experiments, and/or cryopreserved in 10% dimethyl sulfoxide (DMSO)/FBS for future work at concentrations of up to 100 x 10⁶ cells/vial.

2. Flow Cytometry of CLL Cells

1. Label 12 mm x 75 mm round bottomed polystyrene tubes as described in Table 1.
2. In tubes 2 – 5, put one drop of compensation beads and store on ice. Add 1 µL of the appropriate antibody (anti-CD5, CD19, CD23 or CD45, as indicated in Table 1) to tubes 2 – 5, and incubate on ice for 20 min, protected from light by placing tin foil over the ice bucket.
   NOTE: These tubes serve as compensation controls for setting up the flow cytometry template.
3. Put up to 1 x 10⁶ CLL cells into tubes 1, 6 and 7, add 2 mL of FACS buffer (PBS + 2% FBS) to each tube and centrifuge at 300 x g for 5 min at RT to wash the cells. Discard the supernatant and store the tubes containing cell pellets on ice.
   1. Resuspend the cell pellets and add the appropriate combination of antibodies to the cells in tube 7 as instructed in Table 1, in a final volume of 100 µL with FACS buffer. Antibodies are used at an appropriate concentration according to the manufacturer guidelines.
   2. Resuspend the cell pellets in tubes 2 and 6 in 100 µL of FACS buffer.
   3. Incubate the cells on ice, alongside the stained beads in tubes 2 – 5, protected from light for 20 min.
4. After the incubation, add 2 mL of FACS buffer to all the tubes and centrifuge at 300 x g for 5 min at RT to wash the cells. Discard the supernatant and resuspend the bead/cell pellets by gently flicking the tubes.
5. Resuspend tubes 1 – 5 in 100 µL of FACS buffer and place on ice until ready to analyze on the flow cytometer.
6. Dilute the DAPI solution to 0.05-0.2 µg/mL in FACS buffer immediately prior to use. The optimal concentration may vary, and titration is recommended.
7. Resuspend tubes 6 and 7 with 100 µL of diluted DAPI solution and incubate the tubes on ice for a minimum of 5 min to allow the cells to stain.
   NOTE: No further washing is necessary as DAPI must be present in the buffer for dead cells to remain labelled. Once DAPI has been added, cells must be analyzed on the flow cytometer within 4 h.
8. Analyze cells using a flow cytometer.

3. 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 (steps 3.4 & 3.5) or to generate whole cell extract (step 3.6).
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 (step 3.2.3) 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 (step 3.3), the freshly generated cytoplasmic fractions (step 3.4.3), the freshly generated nuclear fractions (step 3.5.3), and the whole cell lysates (step 3.6.3). Pre-chill these microfuge tubes on ice until required.
3. Transfer the cells into individually labelled 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 (step 3.2.1). 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 (step 3.2.2). 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, labelled microfuge tube. This cytoplasmic fraction can be stored at -80 °C until required for further analysis. The remaining pellet contains the nuclear fraction (step 3.5).
      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 (step 3.2.4) 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, labelled 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.
6. Preparation of whole cell lysates (WCL) from CLL cells
   NOTE: The preparation of the whole cell extract can be carried out at the same time as the preparation of the nuclear fractions (step 3.5).
   1. Resuspend the whole cell extract pellets in 100 μL of complete lysis buffer (prepared in step 3.2.4) by pipetting up and down, then add 5 μL of detergent to ensure complete cell lysis. 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 microfuge tube. This whole cell lysate can be stored at -80 °C until required for further analysis.
      NOTE: Avoid repeated freeze/thaw cycles of the samples.

4. Downstream Analysis of Subcellular Fractions

NOTE: In this protocol, analysis of the generated cell fractions was carried out by Western blotting using standard protocols, loading equal cell numbers/lane (equivalent to ~10 µg of protein) for nuclear and cytoplasmic fractions.

1. Quantification of protein trafficking between nuclear and cytoplasmic fractions: Perform quantitative Western blot analysis through quantitation of signal intensity, or densitometry using freely available Western blot analysis software.
2. Importing images: Western blot images generated from different developing instruments must be imported as JPG, PNG or TIFF files. A 16-bit depth RAW file is recommended. To import an image, click the software icon and hover over Import. Then click Third-Party Images. Select the image file and click Open.
3. Displaying the image: In the Image ribbon, click on the Choose button in the Display group. The Adjust Display dialog will open to enable further adjustments if necessary. Implement additional enhancements including Brightness or Contrast using the adjustable sliders on the Image LUTs tab. Use the Curves tab for finer adjustments.
4. Data analysis (channel deselection): Click the Analysis ribbon. To analyze only one channel, deselect the channel(s) not being analyzed. Click on a channel’s Don’t Show Channel thumbnail in the Image LUTs, leaving only the desired channel displayed. Images imported as JPG, PNG or TIFF files may require deselection of multiple unwanted RGB channels.
   1. Adding shapes: To quantify signal intensity, click Add Rectangle to add a rectangle to the image. Click the center of a feature (e.g., a protein band) to place a rectangle around it. Alternatively, to manually draw a shape, choose Draw Rectangle. After adding all the desired shapes, click Select to return the cursor to the selection tool.
      NOTE: Add multiple shapes in a logical order, as the data is sorted by an ID number that is generated sequentially.
   2. Background subtraction: To subtract background noise, click the first button in the Background group and select Median from the drop-down menu. Set the border width to 3 in the Background dialogue and select the segments to be used for the background calculation. When choosing which segments to use, select segments that best represent the image background.
      NOTE: Background noise can affect signal quantification, so it must be subtracted to accurately calculate signal from the shapes of interest.
   3. Trim Signal and Trim Background – OPTIONAL: Files imported in JPG, PNG or TIFF formats may exhibit pixel saturation: highlighted/bright regions within a protein band. Trim Signal and Trim Background (Bkgnd) removes saturated pixels from analysis. To view these values, add Trim Signal and Trim Bkgnd to a table by clicking on the Columns button to the right of the table view.
      NOTE: Pixel saturation may lead to unreliable quantification. Saturated pixels can only be removed if fewer than 5% of pixels within a shape are saturated.
5. Export Data: Click the Shapes tab above the table. For densitometry, values in the Signal column are required. Signal is the sum of the pixel intensity values (Total) for a shape minus the product of the Bkgnd and the Area. Click the Report button. Click Save As or Launch Spreadsheet.
   Signal = Total – (Bkgnd x Area)
   NOTE: The shapes tab provides a table of quantitative values including Signal, Total, Area and Bkgrnd.
6. Quantifying protein expression: Within the saved spreadsheet, calculate normalized expression of the protein of interest for each lane or variable by dividing the obtained Signal for the protein of interest by the Signal for the corresponding protein loading control band.
   NOTE: Comparisons between the amounts of a normalized protein of interest across nuclear and cytoplasmic fractions cannot be directly compared because of the different loading controls used to distinguish nuclear and cytoplasmic fractions. However, comparisons within individual fractions, e.g. following drug treatment, are appropriate.
7. Export image for publication or presentation: Click the Images tab found above the table and then click on the image to be exported. If using the image for a slide presentation or other digital formats, click the software icon, hover over Export and click on Image for Digital Media. Save the image as a JPG, PNG or TIFF file, as required.