Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Summary

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Abstract

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs^1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects^3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.

Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it^8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.

To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome^11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible^12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis¹⁴, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.

In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media^12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

Protocol

1. LysoTracker Staining of Mouse Embryos

1. Generate mouse embryos by placing young (5-6 week old) females into male stud cages. Monitor on following mornings for the appearance of a vaginal plug indicating that mating has occurred. If the female is pregnant, noon on that day is called 0.5 dpc (days post coitus). The procedure presented here is ideal for embryos 7-13 dpc.
2. On the embryonic day of interest, euthanize the female according to approved protocols and remove the uterus. Remove the embryos from the decidua in a 10 cm Petri dish with Hanks BSS (without phenol red). Remove the extra-embryonic membranes and do at least one rinse ....

Discussion

Important hallmarks of apoptosis include the detection of DNA damage with the TUNEL assay, along with the detection of caspase activity (detected with mAbs or a binding molecule such as ZVAD-fmk), the observation of cellular changes, and the presentation of phosphatidylserine (PS) on the membrane surface (detected by Annexin V binding). There are some disadvantages with each of these assays. For example, the terminal transferase used in the TUNEL assay does not penetrate beyond a few cell layers. Also Annexin V can.......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent	Company		Catalogue number
LysoTracker Red DND-99	Invitrogen	#L-7528
Hanks BSS	Invitrogen	14025-076
Paraformaldehyde	EMD	EM-PX0055-3
Vectashield	VECTOR	H-1200
DMEM	Cellgro	10-013-CV
Non-essential amino acids	Cellgro	25-025-CI
Sodium pyruvate	Cellgro	25-000-CI
FBS	Hyclone	SH30071.02
Pen-Strep	Invitrogen	15140-122
b-Mercaptoethanol, 50 mM	Invitrogen	21985-023
LabTek-II Chamber slides (8-well)	Nalge Nunc International	154534
0.1% Gelatin	Millipore	ES-006-B
Dulbecco's PBS (D-PBS)	Cellgro	21-031-CV
			Solution Recipes 4% Paraformaldehyde For 100 ml: Mix 4 g paraformaldehyde, 90 ml H2O, and NaOH (a drop of 2N NaOH). The paraformaldehyde will not go into solution until you have added some NaOH to increase the pH. Stir and heat at 60 °C until all the powder is in solution (~10-20 min). Do not overheat. Add ~10 ml 10x PBS to achieve a final volume of 100 ml. Store at -20 °C in convenient (~10 ml or ~40 ml) aliquots. WARNING: Paraformaldehyde in 'frill' form (compressed small pellets) is less powdery and can therefore be measured outside of a hood. However you should still wear a protective dust mask (N95 at least) during handling. EB Culture Media For 500 ml EB Media: DMEM: 404.5 ml FBS: 75.0 ml L-Glutamine: 5.0 ml Penicillin/Streptomycin: 5.0 ml Non-essential amino acids: 5.0 ml Sodium pyruvate: 5.0 ml β-Mercaptoethanol: 500 μl