Fractionation of neuronal cells into soma and neurite fractions

Fractionation of neuronal cells into cell body and neurite fractions

Materials

Transparent PET transwell membrane (1.0 um pore size) (Corning 353102)

Companion 6-well plates for cell culture inserts (Corning 353502)

Zymo QuickRNA MicroPrep kit (Zymo R1050)

Cell scraper (for example CellTreat 229305)

Razor blades

NuPAGE 4X LDS Sample buffer (Invitrogen NP0008)

Monoclonal beta-actin antibody (Sigma A5441)

Polyclonal Histone H3 antibody (Abcam ab10799)

Matrigel (VWR 47743-706)

Prepare porous membranes

    1. Dilute matrigel to 0.2% in media. Place 1 mL of this solution on the bottom side of transwell membranes. Incubate at 37C for one hour. This is most easily done in a 15cm plate. 

Plate cells

    2. Remove Matrigel solution from membranes.

    3. In each well of 6 well plate, put 4 mL of media.

    4. Put Transwell filter into each well.

    5. Plate 2 mL cell suspesnsion (~1 million cells) onto each filter.

    6. If media change is necessary, allow cells to attach for 1 hr, then replace media above and below membrane.

    7. Allow cells to incubate at 37C for 48 hrs, or longer depending on cell type and desired amount of neurite growth.

Mechanical fractionation of neuronal cells

    8. Remove growth media from above and below the transwell membrane.

    9. Replace media with PBS (2 mL below membrane and 1 mL above membrane).

    10. Pick up transwell membrane and scrape it gently but thoroughly with a cell scraper. Cell bodies will be scraped into the 1 mL PBS that was placed above the membrane.  Be sure to scrape thoroughly over the whole membrane, especially around the junctions of the membrane and the plastic housing.  Forceful manipulation of the membrane can cause it to tear. If this happens, through the membrane away and move on to the next one.

    11. Remove 700 uL of the cell body / PBS solution to a 15 mL Falcon tube on ice.

    12. Tilting the membrane so that the remaining 300 uL of PBS pools at the bottom, scrape the membrane again so that the cell bodies end up in the 300 uL.

    13. Remove the remaining 300 uL into the 15 mL falcon tube.

    14. Turn the membrane over so that the underside is now facing up. 

    15. Using a razor blade, remove the membrane from the plastic housing, staying approximately 2 mm away from the plastic housing. Do not worry about getting all of the membrane that is right next to the plastic as this area is difficult to scrape and may still contain cell bodies.

    16. Using tweezers, put the membrane neurite-side down into a 6 cm plastic dish that contains 500 uL of RNA lysis buffer (Zymo QuickRNA Microprep kit).

    17. Repeat steps 10 through 16 for the remaining membranes.

Preparation of neurites

    18. Once all 6 membranes are in the 6 cm dish, incubate the dish at 4C for 15 minutes with rocking.

    19. Tilting the dish so that all of the liquid collects at the bottom, remove it into a 1.5 mL Eppendorf tube. 

    20. To prepare a sample for Western blot confirmation, take 50 uL of the neurite solution and add to 50 uL of SDS-PAGE loading buffer (Invitrogen NP0008). The RNA lysis buffer contains high amount of guanidine. This can cause the SDS in the loading buffer to precipitate. When this happens, it is not a problem. Before loading the sample on an SDS-PAGE gel, incubate it at 95C for 5 minutes to resolubilize the SDS, then load the sample while it is still hot.

    21. The remaining 450 uL of RNA lysis buffer will be used as the input for RNA isolation.

Preparation of cell bodies

    22. Spin 15 mL Falcon tube containing cell bodies in PBS at 2000 g for 5 min. Remove supernatant, then resuspend in 600 uL ice cold PBS.

    23. To prepare a sample for Western blot confirmation of fractionation efficiency, take 10 uL of resuspended cell bodies and add to 90 uL of SDS-PAGE loading buffer (Invitrogen NP0008).

    24. Of the remaining cell body suspension, 100 uL will be used for RNA isolation. To 100 uL of this suspension, add 350 uL of RNA lysis buffer.  Mix by pipetting up and down.

RNA isolation

    25. You are left with 450 uL of RNA lysis buffer for both the cell body and neurite fractions. Isolate RNA from them using the QuickRNA Microprep Kit (Zymo R1050) according to the manufacturer’s instructions, beginning with the addition of 450 uL (1 volume) of ethanol.

    26. In the final elution of the RNA from the spin column, elute in 10 uL RNase-free water.

    27. Expect between 2 and 10 ug of RNA for the cell body fraction and between 100 and 1000 ng of RNA for the neurite fraction, as quantified by spectroscopy.

Assaying fractionation efficiency by Western blot

    28. Load 5 uL of cell body lysate (step 16) and 15 uL neurite lysate (step 13) on an SDS-PAGE gel. Transfer to nitrocellulose or PVDF membrane.

    29. Probe membrane with antibodies against beta-actin (1:5000) and histone H3 (1:10000) overnight at 4C.

    30. Visualize antibodies using HRP-conjugated secondary antibodies. If the fractionation was efficient, you should expect to see beta-actin and histone H3 signal in the cell body fraction while the neurite fraction should contain only beta-actin signal. For an example of the Western blot of a successful fractionation, see Figure 1C of <Goering et al, eLife 2020>.

Note: The high salt content of the neurite fraction due to the guanidine content of the RNA lysis buffer can often cause these samples to migrate anomalously through the SDS-PAGE gel. The most often results is a “narrowing” of these bands. However, this aesthetic change does not hinder the ability to see protein bands and make conclusions.

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