Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells
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Dorota Sawicka
,
Luiza Chojnacka-Puchta
Abstract
Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank’s solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.
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Articles in the same Issue
- Identification of drought-induced transcription factors in Sorghum bicolor using GO term semantic similarity
- Evaluation of the potential of alkylresorcinols as superoxide anion scavengers and sox-regulon modulators using nitroblue tetrazolium and bioluminescent cell-based assays
- Cyclic phosphatidic acid induces G0/G1 arrest, inhibits AKT phosphorylation, and downregulates cyclin D1 expression in colorectal cancer cells
- Polymorphism of the APEX nuclease 1 gene in keratoconus and Fuchs endothelial corneal dystrophy
- FE65: Roles beyond amyloid precursor protein processing
- Single nucleotide polymorphisms in the STAT3 gene influence AITD susceptibility, thyroid autoantibody levels, and IL6 and IL17 secretion
- Antioxidant effect of a fermented powder of Lady Joy bean in primary rat hepatocytes
- Oncogene-dependent survival of highly transformed cancer cells under conditions of extreme centrifugal force – implications for studies on extracellular vesicles
- Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field
- Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells
- GGeneration of an efficient artificial promoter of bovine skeletal muscle alpha-actin gene (ACTA1) through addition of cis-acting element
