Developing techniques to investigate the transportation of macromolecules between the nucleus and the cytoplasm, and identifying mislocalization of tumor suppressor proteins is essential to gain a deeper understanding of CLL pathogenesis and aid in the development of novel therapies. This protocol provides a fast and efficient method for generating nuclear and cytoplasmic fractions from primary CLL cells. These fractions can be used in downstream experiments including enzyme activity assays, proteomic analysis, and western blotting.
This technique will broaden our understanding of how therapies and micro-environmental signals within tumors alter the shuttling of proteins between the nucleus and cytoplasm in CLL cells and potentially other B cell malignancies. This method can be used to determine the location of proteins and/or attract protein movement as applicable. Perform a detergent gradient on each individual cell line you plan to use to ensure the optimal generation of highly enriched nuclear and cytoplasmic fractions.
Alongside Jodie Hay and Michael Moles, demonstrating this procedure will be Jennifer Cassels, a technician from my laboratory. Obtain peripheral blood samples from previously consented chronic lymphocytic leukemia patients in EDTA blood collection tubes, accompanied by the white cell count. Human blood samples should be regarded as hazardous as they potentially contain blood-borne viruses.
Please ensure that these samples are handled in a class two bio-safety cabinet and that the user is wearing a lab coat and gloves at all times. Dispose of blood-contaminated materials in disinfectants. If the white cell count is equal or greater than 40 million cells per milliliter, dilute the sample at a ratio of one to one with RT CLL wash buffer.
Then aliquot RT density gradient medium into an appropriately sized conical centrifuge tube for the sample. Carefully layer the sample on top of the medium and centrifuge at 400 x g for 30 minutes at RT.Using a plastic Pasteur pipette, gently harvest the white layer of mononuclear cells that collected at the interface of the density gradient medium in CLL wash buffer. Transfer this isolated monolayer into a fresh 50-milliliter conical centrifuge tube.
To wash the cells, add 40 milliliters of CLL wash buffer to this monolayer and centrifuge at 300 x g for 10 minutes at room temperature. Discard the supernatant, flick the bottom of the tube to re-suspend the pellet, and repeat this wash. Discard the supernatant, flick the bottom of the tube again, and then re-suspend the cell pellet in a set volume of CLL wash buffer depending on the size of the pellet.
After counting the cells and after flow cytometry to check the cell purity, place the cells into tissue culture plates at the required cell density ready for stimulation or drug treatment. After stimulation and/or drug treatment, incubation is complete. Transfer the cells into individually labeled 1.5-milliliter microfuge tubes and pellet by centrifuging at 200 x g for five minutes at four degrees Celsius.
After discarding the supernatant, re-suspend the cell pellet in one milliliter of ice-cold PBS with phosphatase inhibitors. Centrifuge at 200 x g for five minutes at four degrees Celsius. Remove the supernatant and keep these whole cell extract pellets on ice for further preparations.
To prepare cytoplasmic fractions, gently re-suspend the cell pellets in 50 microliters of 1x hypertonic buffer. To allow the cells to swell, incubate them on ice for 15 minutes. After a performing a detergent gradient to determine the optimal concentration of detergent to use for a specific cell type, add 0.8 to 2.5 microliters of detergent into each sample and vortex on the highest setting for 10 seconds.
To verify cell lysis, observe the cells under a phase contrast microscope before and after addition of detergent. All cells have a dense, dark nucleus surrounded by the cytoplasm that appears as a bright halo. Once lysed, centrifuge the samples at 14, 000 x g for 30 seconds at four degrees Celsius.
Carefully, without disturbing the pellet, transfer the supernatant into a pre-chilled labeled microfuge tube and store this cytoplasmic fraction at 80 degrees Celsius until further analysis. Ensure the complete removal of the cytoplasmic fraction to prevent contamination of the nuclear fraction. To the remaining pellet, which contains the nuclear fraction, add 50 microliters of complete lysis buffer and re-suspend by pipetting up and down.
To solubilize proteins associated with the nuclear membrane, add 2.5 microliters of detergent, vortex on the highest setting for 10 seconds, and incubate on ice for 30 minutes. Vortex on the highest setting for 30 seconds and centrifuge. Then transfer the supernatant into a pre-chilled labeled microfuge tube and store this nuclear fraction at 80 degrees Celsius until further analysis.
For whole cell lysates, re-suspend the whole cell extract pellets in 100 microliters of complete lysis buffer by pipetting up and down. To ensure complete cell lysis, add five microliters of detergent and incubate on ice for 30 minutes. Vortex on the highest setting for 30 seconds and then centrifuge at 14, 000 x g for 20 minutes at four degrees Celsius.
Transfer the supernatant into a pre-chilled microfuge tube and store this whole cell lysate at 80 degrees Celsius until further analysis. To quantify protein trafficking between nuclear and cytoplasmic fractions, perform quantitative western blot analysis and import the images. To display the image in the Image ribbon, click on the Choose button in the Display group and click on Chemi Channel.
The Choose Display dialog will open to enable further adjustments if necessary. Implement additional enhancements using the adjustable sliders on the image LUT's tab including brightness or contrast. Use the Curves tab for finer adjustments.
For data analysis, first click the Analysis ribbon. To analyze only one channel, deselect the channels not being analyzed by clicking on a channel's Don't Show Channel thumbnail, leaving only the desired channel displayed. To quantify signal intensity, click Add Rectangle to add rectangles to the image.
Click the center of a feature such as a protein band to place a rectangle around it. After adding the desired shapes, click Select to return the cursor to the selection tool. To subtract background noise, click the first button in the Background group and select Median from the dropdown menu.
Set the border width to three in the Background dialog and select the segments that best represent the image background to be used for background calculation. To export the 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 or total for a shape minus the product of the background in the area. Then click the Report button, click Save As or Launch Spreadsheet. To calculate normalized expression of the protein of interest for each plane or variable within the saved spreadsheet, divide the obtained signal for the protein of interest by the signal for the corresponding protein loading control band.
To export the image, 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, click on Image for Digital Media, and then save the image as required. Enrichment of CLL cells with a WCC greater than 40 million per milliliter using density centrifugation enables a high cell recovery.
Analysis of the sample by flow cytometry after gating on FSC and SSC reveal the purity of CLL cells are more than 95%as indicated by the dual surface expression of CLL cell markers, CD19 and CD5. When immuno blots were performed on the resultant fractions of the CLL cell line, Mac-1, and primary CLL cells, fractionation indicated that the optimal detergent level for Mac-1 cells was at one to 60 dilution, compared with one to 30 for primary CLL cells. When subcellular localization of FOXO1 in nuclear and cytoplasmic fractions was determined in Mac-1 and primary CLL cells, generation of highly enriched fractions was shown by the almost exclusive expression of lamin in the nuclear and beta Tubulin in the cytoplasmic fractions.
FOXO1 expression was reduced in the cytoplasm following treatment with AZD 8055, compared to NDC, accompanied by an increase of FOXO1 expression in the nuclear compartment, thus demonstrating protein translocation. After individual immuno blots from five primary CLL samples were quantified within subcellular fractions, AZD 8055 was found to reduce the levels of FOXO1 expression in the cytoplasm and increase the expression in the nucleus. BCR cross-linking increased cytoplasmic FOXO1 expression.
Remember to keep all reagents and samples on ice to prevent protein degradation. The subcellular fractions generated in this procedure can also be used in enzyme activity assays such as a FOXO activity assay, which enables parallels to be drawn between the cellular location of FOXO1 and its DNA binding activity. The development of this technique has enabled us to address the trafficking of specific proteins within CLL cells in response to selective inhibitors and supports our parallel immunofluorescence studies to generate a robust and quantifiable dataset.
