Application of density gradient centrifugation for separation of eosinophilic and basophilic hemocytes from Mytilus edulis and characterization of both cell groups

Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 × 10(6) and 5.9 × 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

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Characterization of the Impact of Density Gradient Centrifugation on the Profile of the Pig Sperm Transcriptome by RNA-Seq

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.668158 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yu Lian ◽

Marta Gòdia ◽

Anna Castello ◽

Joan Enric Rodriguez-Gil ◽

Sam Balasch ◽

...

Keyword(s):

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Principal Component ◽

Rna Seq ◽

Protein Coding ◽

The Impact ◽

Germinal Cells ◽

Rna Levels

RNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPureTM on porcine sperm. Four boar ejaculates were purified with BoviPureTM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high—although not complete—similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs.

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Cytomorphologic Characterization of Cell Subsets Isolated by Density Gradient Centrifugation from Tumor Effusions of Ovarian Endometrioid Carcinoma

Tumori Journal ◽

10.1177/030089169408000410 ◽

1994 ◽

Vol 80 (4) ◽

pp. 290-294 ◽

Cited By ~ 2

Author(s):

Julia K. Bar ◽

Antonina Harłozińska ◽

Ewa Sobańska ◽

Mieczysław Cislo

Keyword(s):

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Malignant Cell ◽

Endometrioid Carcinoma ◽

Cellular Composition ◽

Poorly Differentiated ◽

Centrifugation Technique ◽

Cell Subpopulations

Aims Cytomorphologic characterization of tumor cell subsets, according to the stage of pathologic differentiation, and comparison of cellular composition in tumor cyst and ascitic fluids were carried out on individual patients with ovarian endometrioid carcinoma. Methods A density gradient centrifugation technique was applied to fractionate the cells from tumor effusions. Results The enrichment of cell forms representing individual stages of pathologic differentiation by gradient centrifugation facilitated their cytomorphologic characterization. According to cytomorphologic features, 5 discrete cell subpopulations were identified and catalogued. The cellular composition of tumor cyst and ascitic fluids in individual patients was similar, but the number of fractions and percentage of cell subsets differed. Conclusions The estimation of precise cytomorphologic criteria for cell forms in tumor effusions facilitated the cytologic diagnosis of ovarian endometrioid carcinoma. The possibility to concentrate poorly differentiated, frankly malignant cell subsets in low densities could significantly improve the diagnosis of tumor effusions.

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