Materials and Reagents

Click-iT^® Cell Reaction Buffer Kit (Life Technologies, Invitrogen^TM, catalog number: C10269 )
EdU (Life Technologies, Invitrogen^TM, catalog number: A10044 )
Alexa Fluor^® 488 azide (Life Technologies, Invitrogen^TM, catalog number: A10266 )
OR Alexa Fluor^® 594 azide (Life Technologies, Invitrogen^TM, catalog number: C10270 )
4% Paraformaldehyde (PFA) in PBS
Deionized water (ddH₂O)
Poly-L-Lysine (Sigma-Aldrich, catalog number: P8920 )
TEFLON printed slides 8-well, 6 mm diameter (Electron Microscopy Sciences)
Vectashield^® mounting medium with DAPI (Vector Laboratories, catalog number: H-1200 )
Nail polish
EdU solution (molecular weight of EdU: 252.22 g/mol) (see Recipes)
0.1 M Glycine in 1x PBS (see Recipes)

Equipment

A humid chamber (a box with moistened paper tower; can protect from light and at least 8 x 10 cm²)
Microscope Coverslips, 22 x 50 cm² (Fisherbrand^®)
Centrifuge
28 °C 5% CO₂ Cell culture incubator
Fluorescent microscope

Procedure

I. EdU labeling of trypanosome cells

Notes:
1)This protocol has not been used for the bloodstream form of trypanosomes yet. However, it is supposed to work well for the bloodstream form of trypanosomes too.
2)From step 3 to the end of the protocol, operate at room temperature (RT).
3)From step 9, please protect from light by covering the box with lid or the eppendorf tubes with foil paper. In steps 7, 8, and 10, use a 100 or 200 μl pipette to remove solution from each well.

Culture procyclic trypanosomes in 10 ml SDM-79 medium supplemented with 10% FBS at 28 °C, 5% CO₂ incubator.
Add EdU solution to cell culture at final concentration of 50 μM to 100 μM and continue to incubate at the same condition as step 1 for 30 min to 3 h as planned.
Trypanosome cells are then pelleted at 700 x g for 5 min.
Wash the cells with 1 ml 1x PBS once and resuspend cells in 1x PBS at around 2 x 10⁷ cells/ml.
Coat the 8-well glass slide with poly-L-lysine (0.1% w/v in H₂O).
Add 25 μl cell suspension onto each well in a humid chamber (see Equipment 1) and seat for 5 min.
Remove cell suspension and add 40 μl 4% paraformaldehyde to fix cells for 5 min.
Remove fixatives and wash cells twice with 50 μl 0.1 M Glycine for 5 min.
Prepare Click-iT reaction cocktail (make cocktail fresh and keep dark).
For 200 μl (1 slide or 8 wells, 25 μl/well):
Component A: 1x TBS 176 μl
Component B: 100 mM CuSO₄ 4 μl
Component C: Buffer additive    20 μl
1 mM Alexa-488-azide     1 μl
(OR 1 mM Alexa-594-azide    1 μl)
Remove the wash buffer from each well at step 8 and add 25 μl reaction cocktail per well and incubated for 1 h at RT. Protect from light.
After 1 h incubation, remove the reaction cocktail, wash twice with 50 μl 1x PBS. Protect from light.
Mount slides with Vectashield Mounting Medium with DAPI (1.5 μg/ml), seal with nail polish and seat for 15~30 min. Protect from light.
Examine by Fluorescent Microscopy (using 63x or 100x objective lens) (Figure 1).

Figure 1. EdU labeling of procyclic form of trypanosome cells. DNA synthesis was measured by adding 100 μM EdU to the culture medium for 1 h before harvest. Cells were then adhered to a poly-L-lysine coated 8-well slide and EdU was detected with Alexa-488-azide followed by the counterstaining of DNA and nucleus with 1.5 μg/ml DAPI. The inset in the EdU panel is the enlarged EdU labeling image of the cell on the right in the Phase panel. N, nucleus; k, kDNA. Red, DAPI; Green, EdU. Bar, 5 μm.

II. EdU labeling of kDNA networks isolated from trypanosomes

Notes:
1)From step 3 to the end of the protocol, operate at RT (room temperature).
2)From step 7, please protect from light by covering the box with lid or the eppendorf tubes with foil paper.
3) In steps 8 and 9, please use a 100 or 200 μl pipette to remove solution from each well.

Culture the procyclic trypanosomes in SDM-79 medium supplemented with 10% FBS at 28 °C, 5% CO₂ incubator. For kDNA isolation, prepare more than 1 x 10⁸ cells in total.
Add EdU solution to cell culture at 50 μM to 100 μM and continue to incubate for 30 min to 3 h as planned.
Isolate kDNA networks from ≥ 5 x 10⁷ trypanosome cells.
Coat the 8-well glass slide with 1:50 diluted poly-L-lysine in ddH₂O.
Mix 1 μl isolated kDNA networks with 19 μl 1x PBS (add 1x PBS in each well first and then mix kDNA with 1x PBS on the well).
Note: Before doing EdU labeling of isolated kDNA networks, it is suggested to examine kDNA networks abundance by DAPI staining only by following steps 5, 6, 10 and 11 (at least 15 kDNA networks in each field to continue).
Allow kDNA networks seat on the slide (poly-L-lysine coated) for 30 min at room temperature in humid chamber (see Equipment 1).
Prepare Click-iT reaction cocktail (make cocktail fresh and keep dark).
For 200 μl (1 slide or 8 wells, 25 μl/well):
Component A: 1x TBS     176 μl
Component B: 100 mM CuSO₄ 4 μl
Component C: Buffer additive    20 μl
1 mM Alexa-488-azide     1 μl
(OR 1 mM Alexa-594-azide      1 μl)
Remove kDNA and 1x PBS mixture from each well and add 25 μl reaction cocktail per well and incubated for 1 h at RT. Protect from light.
After 1 h incubation, remove the reaction cocktail, wash twice with 50 μl 1x PBS. Protect from light.
Mount slides with Vectashield Mounting Medium with DAPI (1.5 μg/ml), and seal with nail polish, and seat for 15~30 min. Protect from light.
Examine by Fluorescent Microscopy (using 100x objective lens) (Figure 2).

Figure 2. EdU labeling of isolated kDNA network from trypanosome cells. Networks were isolated from EdU-labeled cells and adhered to an 8-well glass slide and incorporated EdU was then detected with Alexa-488-azide (in green) and networks were stained with 1.5 μg/ml DAPI (in red). Arrows, a unit-sized pre-replication kDNA; Arrowhead, a double-sized post-replication kDNA.

Recipes

EdU solution (molecular weight of EdU: 252.22 g/mol)
Dissolve 12.16 mg EdU in 1 ml ddH2O for 50 mM EdU solution
Dissolve 12.16 mg EdU in 1 ml ddH2O for 50 mM EdU solution
1x PBS
Dissolve the following in 800 ml distilled H₂O
8 g of NaCl
0.2 g of KCl
1.44 g of Na₂HPO₄
0.24 g of KH₂PO₄
Adjust pH to 7.4; then adjust volume to 1 L with additional distilled H₂O. Sterilize by autoclaving.

Acknowledgments

I thank all lab members of Paul Englund and Robert Jensen for helpful discussions. This work was supported by NIH grant AI058613.

References

Wang, J., Englund, P. T. and Jensen, R. E. (2012). TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance. Mol Microbiol 83(3): 471-485.