Sam’s trypanosome protocols
Making procyclic cytoskeletons retaining the nuclear DNA integrity
Notes
NP40 extracts membranous material and cytoplasmic material, leaving only
cytoskeleton associated material behind.
Trypanosomes have a tough, microtubule based cytoskeletal ‘cage’, which is easily
visible by phase contrast microscopy.
It is vital that at no point in the procedure do the cytoskeletons dry. If this happens,
the nucleus loses integrity and is no longer visible by phase and DAPI staining as a
discrete structure.
The shape of the kinetoplast is difficult to retain, and tends to take on a more rounded
form than in live cells.
If cells are not properly washed, they will not stick to the slide. This is because
residual FCS from the media will block the protein binding capacity of the glass.
Prepare in advance:
 Pre-chilled (-20C) methanol
 vPBS (137mM NaCl, 3mM KCl, 16mM Na2HPO4, 3mM KH2PO4 , 46mM
sucrose, 10mM glucose, pH 7.6)
 PEME (100mM PIPES, 2mM EGTA, 0.1mM EDTA, 1mM MgSO4, pH 6.9)
 Glass slides with a large square well drawn using a fat pen
 Mount (1% DABCO, 90% glycerol, 466l H2HPO4, 34l NaH2PO4, 1 5g.ml-1 DAPI)
Protocol
1. Cells should be happy and healthy in mid-log (5x106 – 1x107 cells per ml for
procyclic forms)
2. Pipette 1ml of cell culture into a 1.5ml eppendorf
3. Centrifuge for 3 minutes at 1000g
4. Remove as much supernatant as possible (this is most easily done using the
aspirator, but for small numbers of samples can also be done by pipetting)
5. Re-suspend cell pellet in 1ml of room temp vPBS
6. Repeat centrifugation
7. Re-suspend cells in 500l of room temp vPBS
8. Add 50l of cells to a well on the glass slide, spread using a yellow pipette tip
9. Watch the live cells sticking to the glass slide under the microscope
10. When the stuck cells are at the desired density (takes between 1-5 minutes):
a. remove excess vPBS by pipetting
b. add 50l 0.5% NP40 in PEME for 10 seconds
c. gently tap off excess liquid
d. dunk in -20C methanol for 20 minutes
11. Rehydrate in 1xPBS
12. Cytoskeletons can remain in cold PBS and still retain good morphology,
antigenicity and fluorescence for several weeks