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An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.
Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.
In animal development, hundreds, thousands, millions, billions, or even trillions of cell divisions are required to form the adult organism. The cell cycle, the set of cellular events composed of G1 (gap), S (synthesis), G2 (gap), and M (mitosis) define the series of events that are executed each cell division. The cell cycle is dynamic and best appreciated in real time, which can be technically difficult. The techniques presented in this protocol allow one to make the measurements of the phases and timing of the cell cycle from still images.
Labeling with nucleoside analogs such as 5-ethynyl-2'-deoxyuridine (EdU) or 5-bromo-2'-deox....
1. Preparation of EdU-labeled Bacteria
Since DNA synthesis is required to incorporate EdU, one can conclude that EdU-labeled nuclei underwent S-phase during the EdU-labeling time window. One may interpret the nuclei that label in a 30 min feeding with EdU labeled bacteria as nuclei in S-phase at the time of dissection. Nuclei that label in a longer continuous EdU feeding experiment may have labeled early in the time window and since left S-phase, or may have labeled in the late part of the EdU time window. EdU signal co-locali.......
Preparation of EdU-labeled bacteria (step 1) is critical for this protocol, and the first point for troubleshooting. Wild-type young adult hermaphrodites label very reliably in a 4 h EdU-pulse, making this a useful control for every new batch of EdU-labeled bacteria. Additionally, intact EdU-labeled bacteria that enter the intestine (in older animals or certain pharynx/grinder defective mutants) will label with click chemistry and appear as bright oblong puncta in the gut. An alternative technique for labeling hermaphrod.......
We are grateful to the E. coli stock center for MG1693; Wormbase; the Caenorhabditis Genetics Center which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40OD010440) for strains; Zach Pincus for statistical advice; Aiping Feng for reagents; Luke Schneider, Andrea Scharf, Sandeep Kumar, and John Brenner for training, advice, support, and helpful discussion; and the Kornfeld and Schedl labs for feedback on this manuscript. This work was supported in part by National Institutes of Health [R01 AG02656106A1 to KK, R01 GM100756 to TS] and a National Science Foundation predoctoral fellowship [DGE-1143954 and DGE-....
Name | Company | Catalog Number | Comments |
E. coli MG1693 | Coli Genetic Stock Center | 6411 | grows fine in standard unsupplemented LB |
E. coli OP50 | Caenorhabditis Genetics Center | OP50 | |
Click-iT EdU Alexa Fluor 488 Imaging Kit | Thermo Fisher Scientific | C10337 | |
5-Ethynyl-2′-deoxyuridine | Sigma | 900584-50MG | or use EdU provided in kit |
Glucose | Sigma | D9434-500G | D-(+)-Dextrose |
Thiamine (Vitamin B1) | Sigma | T4625-5G | Reagent Grade |
Thymidine | Sigma | T1895-1G | BioReagent |
Magnesium sulfate heptahydrate | Sigma | M1880-1KG | MgSO4, Reagent Grade |
Sodium Phosphate, dibasic, anhydrous | Fisher | BP332-500G | Na2HPO4 |
Potassium Phosphate, monobasic | Sigma | P5379-500G | KH2PO4 |
Ammonium Chloride | Sigma | A4514-500G | NH4Cl, Reagent Plus |
Bacteriological Agar | US Biological | C13071058 | |
Calcium Chloride dihydrate | Sigma | C3881-500G | CaCl |
LB Broth (Miller) | Sigma | L3522-1KG | Used at 25g/L |
Levamisole | Sigma | L9756-5G | 0.241g/10ml |
Phosphate buffered saline | Calbiochem Omnipur | 6506 | homemade PBS works just as well |
Tween-20 | Sigma | P1379-500ML | |
16% Paraformaldehyde, EM-grade ampules | Electron Microscopy Sciences | 15710 | 10ml ampules |
100% methanol | Thermo Fisher Scientific | A454-1L | Gold-label methanol is critical for proper morphology with certain antibodies |
Goat Serum | Gibco | 16210-072 | Lot 1671330 |
rabbit-anti-WAPL-1 | Novus biologicals | 49300002 | Lot G3048-179A02, used at 1:2000 |
mouse-anti-pH3 clone 3H10 | Millipore | 05-806 | Lot#2680533, used at 1:500 |
goat-anti-rabbit IgG-conjugated Alexa Fluor 594 | Invitrogen | A11012 | Lot 1256147, used at 1:400 |
goat-anti-mouse IgG-conjugated Alexa Fluor 647 | Invitrogen | A21236 | Lot 1511347, used at 1:400 |
Vectashield antifade mounting medium containing 4',6-Diamidino-2-Phenylindole Dihydrochloride (DAPI) | Vector Laboratories | H-1200 | mounting medium without DAPI can be used instead, following a separate DAPI incubation |
nail polish | Wet n Wild | DTC450B | any clear nail polish should work |
S-medium | various | see wormbook.org for protocol | |
M9 buffer | various | see wormbook.org for protocol | |
M9 agar | various | same recipe as M9 buffer, but add 1.7% agar | |
Nematode Growth Medium | various | see wormbook.org for protocol | |
dissecting watch glass | Carolina Biological | 42300 | |
Parafilm laboratory film | Pechiney Plastic Packaging | PM-996 | 4 inch wide laboratory film |
petri dishes | 60 mm diameter | ||
Long glass Pasteur pipettes | |||
1ml centrifuge tubes | MidSci Avant | 2926 | |
Tips | |||
Serological pipettes | |||
500 mL Erlenmyer flask | |||
Aluminum foil | |||
25G 5/8” needles | BD PrecisionGlide | 305122 | |
5ml glass centrifuge tube | Pyrex | ||
Borosilicate glass tubes 1ml | |||
glass slides | |||
no 1 coverslips 22 x 40 mm | no 1.5 may work, also | ||
37 °C Shaker incubator | |||
Tabletop Centrifuge | |||
Clinical Centrifuge | IEC | 428 | with 6 swinging bucket rotor |
Mini Centrifuge | |||
20 °C incubator | |||
4 °C refrigerator | |||
-20 °C freezer | |||
Observer Z1 microscope | Zeiss | ||
Plan Apo 63X 1.4 oil-immersion objective lens | Zeiss | ||
Ultraview Vox spinning disc confocal system | PerkinElmer | Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy |
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