The number of apoptotic cells in the gonads of day-2 animals was assessed by scoring the number of SYTO12 labeled cells in the gonad. SYTO12 (Molecular Probes) staining was performed as previously described [81 (link)]. In short, to obtain an estimate of the relative numbers of corpses in different genetic backgrounds, 2-day adult animals were stained with SYTO 12 (Molecular Probes, Eugene, OR), a vital dye that preferentially stains apoptotic germ cells. Animals were stained by incubating them in a 33 μM aqueous solution of SYTO 12 supplemented with OP50 for 4–5 hours at 25°C. Animals were transferred back to new seeded plates to allow stained bacteria to be purged from the gut. After 30–60 minutes, animals were mounted on agarose pads and inspected using a Nikon eclipse 90i, equipped with standard epifluorescence filters and Nomarski optics. Only animals that stained brightly were scored.
Syto 12
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYTO 12 is a nucleic acid stain for fluorescence microscopy. It is a cell-permeant nucleic acid stain that exhibits enhanced fluorescence upon binding to RNA or DNA.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions)
Bongkrekic acid, by Merck Group (1 mentions)
4 4 diisothiocyanatostilbene 2 2 disulfonic acid disodium salt hydrate, by Merck Group (1 mentions)
Ecl plus detection kit, by Merck Group (1 mentions)
Phospho mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Acridine orange, by Dingguo (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
C elegans rnai v1.1 feeding library, by Thermo Fisher Scientific (1 mentions)
Acridine orange, by Thermo Fisher Scientific (1 mentions)
12 protocols using syto 12
1
Quantification of Apoptotic Germ Cells
2
Quantifying Germline Apoptotic Corpses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The number of apoptotic corpses in the germ line was estimated using the vital dye SYTO-12 (Invitrogen): worms were washed in M9, incubated in 200 μL of 33 μM SYTO-12 in M9 for 5 h at room temperature in the dark, destained for 1 h in the dark on an NGM plate freshly seeded with OP50, and scored live while paralyzed in levamisole.37 (link)
3
Quantifying Germ Cell Number and Apoptosis in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify germ cell number worms were stained using 6′-diamino-2-phenylindole hydrochloride (DAPI) [50 (link)]. The number of apoptotic corpses in germlines was estimated using the vital dye SYTO 12 [22 (link)], or using CED-1::GFP [25 (link)].
To quantify germ cell number worms were stained using 6′-diamino-2-phenylidole hydrochloride (DAPI) as previously described [50 (link)]. Worms were fixed in ice-cold methanol for 5 min, washed with M9, and incubated in 500 ng/ml DAPI solution in the dark for 30 min. Worms were then washed once more with M9 and mounted on slides for imaging. DAPI-stained worms were imaged at 200x to obtain an image of the entire visible gonad arm. The open source Image J software (NIH image) with the ITCN plug-in was used to quantify the number of germ cells per gonad arm.
The number of apoptotic corpses in live worms was estimated using the vital dye SYTO 12 (Molecular Probes), as previously described [22 (link)]. Worms were incubated in the dark in 33 μM SYTO 12 for 4 hrs. Worms were then transferred to a freshly seeded OP50 plate for an hour for the stain to be expelled from the gut. Worms were placed on slides and SYTO 12-positive cells, viewed at 200x magnification, were counted manually. Alternatively, worms containing CED-1::GFP which is expressed in the gonad sheath was also used to count engulfed apoptotic cells [25 (link)].
To quantify germ cell number worms were stained using 6′-diamino-2-phenylidole hydrochloride (DAPI) as previously described [50 (link)]. Worms were fixed in ice-cold methanol for 5 min, washed with M9, and incubated in 500 ng/ml DAPI solution in the dark for 30 min. Worms were then washed once more with M9 and mounted on slides for imaging. DAPI-stained worms were imaged at 200x to obtain an image of the entire visible gonad arm. The open source Image J software (NIH image) with the ITCN plug-in was used to quantify the number of germ cells per gonad arm.
The number of apoptotic corpses in live worms was estimated using the vital dye SYTO 12 (Molecular Probes), as previously described [22 (link)]. Worms were incubated in the dark in 33 μM SYTO 12 for 4 hrs. Worms were then transferred to a freshly seeded OP50 plate for an hour for the stain to be expelled from the gut. Worms were placed on slides and SYTO 12-positive cells, viewed at 200x magnification, were counted manually. Alternatively, worms containing CED-1::GFP which is expressed in the gonad sheath was also used to count engulfed apoptotic cells [25 (link)].
4
Quantifying Apoptosis in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult nematodes were suspended in M9 solution and stained by incubation with 33 µM SYTO-12 (Molecular probes) for 1 h and 30 min at room temperature in the dark. The worms were then transferred to seeded plates to allow stained bacteria to be purged from the gut. After 30 min, the animals were mounted on 2% agarose pads in 2 mM levamisole21 (link). The estimation of apoptotic levels for each genotype was calculated as the average number of apoptotic nuclei per gonadal arm22 (link).
5
Investigating MAPK Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MAPK Family Antibody Sampler Kit (#9926) and Phospho-MAPK Family Antibody Sampler Kit (#9910) were purchased from Cell Signaling Technology (Beverly, MA). Anti-CEP-1 (cC-18) (sc-135460) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Paraquat (PQ), Cyclosporin A (CsA), Bongkrekic acid (BA) and 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) were from Sigma. N-acety-L-cysteine (NAC) was purchased from TCI (Shanghai, China). Total Antioxidant Capacity Assay Kit with the ABTS Method (S0119), Fluo-3 AM (S1056), Hoechst 33342 (C1022), Mitochondrial Membrane Potential Assay Kit with JC-1 (C2006), Enhanced BCA Protein Assay Kit (P0010S) and Superoxide dismutase (SOD, S0088) were obtained from Beyotime (Shanghai, China). Acridine orange (AO) was from Dingguo Changsheng Biotechnology (Beijing, China). H2DCF-DA (D399), MitoSox (M36008), Mitotracker red (M7512), ATP determination kit (A22066) and SYTO 12 (S7574) were purchased from Molecular Probes (Eugene, Oregon, USA). PVDF membranes and ECL plus detection kit were obtained from Millipore (Bedford, MA, USA).
6
Apoptosis Quantification in Gonads
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
7
Quantifying Apoptosis in Nematodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adult nematodes were suspended in M9 solution and stained by incubation with 33 µM SYTO-12 (Molecular probes) for 1 hr and 30 min at room temperature in the dark. The worms were then transferred to seeded plates to allow stained bacteria to be purged from the gut. After 30 minutes, the animals were mounted on 2% agarose pads in 2 mM levamisole. The estimation of apoptotic levels for each genotype was calculated as the average number of apoptotic nuclei per gonadal arm17 (link),37 (link).
8
Quantifying Germ Cell Apoptosis in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For single time-point experiments, the number of apoptotic germ cells was scored in day-2 animals stained with SYTO12 (Molecular Probes) as previously described [6] (link). For time course experiments, the number of SYTO12-labeled apoptotic corpses per gonad arm was scored in animals from day-0 (L4) to day-3 of adulthood. Where indicated, the average number of apoptotic corpses was normalized to the number of mitotic germ cells within the proliferative zone of the gonads, determined by section analysis of DAPI-stained gonads.
9
Staining and Scoring C. elegans Corpses
10
Apoptosis Detection in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The response to ascr#10 as an increase in apoptotic cells was determined in three ways. Animals were stained with SYTO12 (Invitrogen) using the protocol of Gumienney et al. (26 (link)). An alternative stain, acridine orange (Invitrogen) was also used to monitor cell deaths as described by Lant and Derry (72 (link)). Both protocols require the hermaphrodites to take up the dye and, later, to consume unstained bacteria to clear the stain from the intestine. Since the worms are alive during the staining protocol, staining began in the morning and finished in the afternoon of the day specified. In addition, the increase of apoptotic cells when exposed to ascr#10 was observed in a strain carrying the CED-1::GFP transgene. This strain cannot be used for quantifying the number of apoptotic corpses, however, because it has been noted that not all cells surrounded by CED-1::GFP go on to form apoptotic corpses (73 (link)). All experiments were processed with paired controls.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!