Separation and characterization of Leydig cells and macrophages from rat testes

1991 ◽  
Vol 130 (3) ◽  
pp. 357-365 ◽  
Author(s):  
G. Dirami ◽  
L. W. Poulter ◽  
B. A. Cooke
Keyword(s):  
Monoclonal Antibody ◽  
Density Gradient ◽  
Leydig Cells ◽  
Magnetic Beads ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Velocity ◽  
Average Density ◽  
Centrifugal Elutriation ◽  
Percoll Gradients

ABSTRACT A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1–F8) (9 to 74 ml/min at 386 g). Each of these fractions was then further purified by density gradient centrifugation on 0–90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1–F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11–20%) were found in fractions F1–F3 (average density 1·045 g/ml), containing 11–37% Leydig cells. Less than 3% of the cells in fraction F4–F8 (average density 1 ·075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated-and Percoll-purified fractions F4–F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for 125I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35·7 mm/h-g) from fractions F7 and F8 were approximately twofold more responsive to LH (3·3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20·7 mm/h-g). The elutriated and Percoll-purified cells (corresponding to fractions F4–F8) were further purified by incubation with magnetic beads coated with a macrophage monoclonal antibody; this yielded very pure Leydig cells containing <0·3% macrophages. The incubation temperature (room temperature or 4 °C) during the purification with magnetic beads did not affect the degree of purity or the responsiveness of the Leydig cells to LH. The removal of the remaining macrophages with magnetic beads did not have any significant effect on the Leydig cell responsiveness to LH. It was concluded that Leydig cells purified by elutriation and density gradient centrifugation are heterogeneous with respect to their sedimentation velocities and responses to LH; the higher the sedimentation velocity, the higher is their capacity to respond to LH. Leydig cells free from macrophages can be prepared by further purification using magnetic beads coated with a macrophage monoclonal antibody. Journal of Endocrinology (1991) 130, 357–365

scholarly journals Macromolecular organization of saliva: identification of ‘insoluble’ MUC5B assemblies and non-mucin proteins in the gel phase

2000 ◽  
Vol 351 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Claes WICKSTRÖM ◽  
Cecilia CHRISTERSSON ◽  
Julia R. DAVIES ◽  
Ingemar CARLSTEDT
Keyword(s):  
Monoclonal Antibody ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Secretory Component ◽  
Significant Fraction ◽  
Guanidinium Chloride ◽  
Whole Saliva ◽  
Gel Phase ◽  
Macromolecular Organization

Stimulated human submandibular/sublingual (HSMSL) and whole saliva were separated into sol and gel phases and mucins were isolated by density-gradient centrifugation in CsCl/4M guanidinium chloride. MUC5B and MUC7 were identified using anti-peptide antisera raised against sequences within the MUC5B and MUC7 apoproteins respectively. MUC7 was found mainly in the sol phase of both HSMSL and whole saliva, but some MUC7 was consistently present in the gel phase, suggesting that this mucin may interact with the salivary gel matrix. In HSMSL saliva, MUC5B was found in the gel phase; however, most of the material was ‘insoluble’in guanidinium chloride and was only brought into solution by reduction. In whole saliva, the MUC5B mucin was present both in the sol and gel phases although some material was again ‘insoluble’. Rate-zonal centrifugation of whole saliva showed that MUC5B mucins in the sol phase were smaller than those in the gel phase, suggesting differences in oligomerization and/or degradation. Antibodies against IgA, secretory component, lysozyme and lactoferrin were used to study the distribution of non-gel-forming proteins in the different phases of saliva. The majority of these proteins was found in the sol phase of both HSMSL and whole saliva. However, a significant fraction was present in the gel phase of whole saliva, suggesting a post-secretory interaction with the salivary gel matrix. A monoclonal antibody against a parotid salivary agglutinin was used to show that this protein is present mainly in the gel phase of both whole saliva and parotid secretion.

ISOLATION OF RAT LEYDIG CELLS BY DENSITY GRADIENT CENTRIFUGATION

1982 ◽  
Vol 92 (2) ◽  
pp. 293-NP ◽  
Author(s):  
J. S. GALE ◽  
J. ST J. WAKEFIELD ◽  
H. C. FORD
Keyword(s):  
Density Gradient ◽  
Binding Sites ◽  
Leydig Cells ◽  
Interstitial Cell ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Collagenase Treatment

A rapid method for preparing Leydig cells from rat testes is described. An interstitial cell suspension, prepared by collagenase treatment of decapsulated testes, was centrifuged for 10 min over a cushion of 60% (v/v) Percoll to remove red blood cells, and then centrifuged for 20 min in a 0–60% linear density gradient of Percoll. Seventy-four per cent of the cells present in that fraction of the gradient comprising 35–50% Percoll were Leydig cells; the yield from each testis was about 1·5 × 106 cells. The Leydig cells appeared viable, excluded Trypan blue, possessed high-affinity binding sites for human chorionic gonadotrophin (hCG) and synthesized increased quantities of testosterone in response to hCG. The cells could be stored overnight in 20% (v/v) glycerol at −20 °C, with only minimal effect on the specific activities of a number of enzymes used as markers of subcellular components. Testosterone production in vitro by the cells after storage for 20 h was greater than that of hCG-stimulated fresh cells and was not further increased by hCG.

ISOLATION OF HIGHLY PURIFIED LEYDIG CELLS BY DENSITY GRADIENT CENTRIFUGATION

Endocrinology ◽  
1977 ◽  
Vol 101 (2) ◽  
pp. 639-642 ◽  
Author(s):  
P. Michael Conn ◽  
Tsuneo Tsuruhara ◽  
Maria Dufau ◽  
Kevin J. Catt
Keyword(s):  
Density Gradient ◽  
Leydig Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation

scholarly journals Failure of autologous marrow reconstitution after cytolytic treatment of marrow with anti-Ia monoclonal antibody

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 819-822 ◽  
Author(s):  
J Szer ◽  
HJ Deeg ◽  
FR Appelbaum ◽  
R Storb
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Density Gradient ◽  
Bone Marrow Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Autologous Bone Marrow Transplantation ◽  
Hematopoietic Stem ◽  
Rabbit Complement ◽  
Marrow Cells

Abstract Hematopoietic stem cell toxicity of the murine monoclonal antibody 7.2, recognizing Ia-like antigens on canine cells, was tested in an autologous bone marrow transplantation model. Dogs were given 9.2 Gy of total body irradiation followed by the infusion of autologous marrow treated by one of two methods to remove Ia+ cells. In six dogs, the marrow cells were pelleted, treated with antibody 7.2 (1:1,000) and rabbit complement (1:4), resuspended in culture medium, and infused. All six dogs had prompt and sustained engraftment surviving greater than 26 days. Indirect immunofluorescence showed, however, that the depletion of Ia+ cells was incomplete. Four dogs received marrow cells first separated by density gradient centrifugation and then treated with an excess of antibody 7.2 and two cycles of undiluted rabbit complement. None of these dogs, surviving 17 to 22 days, had sustained engraftment. With antibody 7.2 used as the marker, only one dog had detectable residual Ia+ cells (0.9%) after treatment. Dogs receiving marrow cells obtained by density gradient centrifugation without additional manipulation, or with subsequent treatment with complement only or with complement and an antibody (DT-2) directed at a subpopulation of T cells, engrafted promptly and completely. We conclude that Ia+ bone marrow cells are essential for the successful engraftment of transplanted marrow in dogs.

Separation of viable microfilariae free of blood cells on Percoll gradients

1984 ◽  
Vol 58 (1) ◽  
pp. 69-70 ◽  
Author(s):  
R. Chandrashekar ◽  
U. R. Rao ◽  
G. R. Rajasekariah ◽  
D. Subrahmanyam
Keyword(s):  
Density Gradient ◽  
Blood Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Brugia Pahangi ◽  
Percoll Gradients ◽  
Reproducible Method ◽  
Dipetalonema Viteae

AbstractA consistent and reproducible method is described for isolating pure populations of microfilariae of Litomosoides carinii, Brugia pahangi, B. malayi and Dipetalonema viteae, free of cells, from blood, by density gradient centrifugation on Percoll in 0.25M sucrose. The recovery of the microfilariae was 85 to 97%.

scholarly journals Failure of autologous marrow reconstitution after cytolytic treatment of marrow with anti-Ia monoclonal antibody

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 819-822
Author(s):  
J Szer ◽  
HJ Deeg ◽  
FR Appelbaum ◽  
R Storb
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Density Gradient ◽  
Bone Marrow Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Autologous Bone Marrow Transplantation ◽  
Hematopoietic Stem ◽  
Rabbit Complement ◽  
Marrow Cells

Hematopoietic stem cell toxicity of the murine monoclonal antibody 7.2, recognizing Ia-like antigens on canine cells, was tested in an autologous bone marrow transplantation model. Dogs were given 9.2 Gy of total body irradiation followed by the infusion of autologous marrow treated by one of two methods to remove Ia+ cells. In six dogs, the marrow cells were pelleted, treated with antibody 7.2 (1:1,000) and rabbit complement (1:4), resuspended in culture medium, and infused. All six dogs had prompt and sustained engraftment surviving greater than 26 days. Indirect immunofluorescence showed, however, that the depletion of Ia+ cells was incomplete. Four dogs received marrow cells first separated by density gradient centrifugation and then treated with an excess of antibody 7.2 and two cycles of undiluted rabbit complement. None of these dogs, surviving 17 to 22 days, had sustained engraftment. With antibody 7.2 used as the marker, only one dog had detectable residual Ia+ cells (0.9%) after treatment. Dogs receiving marrow cells obtained by density gradient centrifugation without additional manipulation, or with subsequent treatment with complement only or with complement and an antibody (DT-2) directed at a subpopulation of T cells, engrafted promptly and completely. We conclude that Ia+ bone marrow cells are essential for the successful engraftment of transplanted marrow in dogs.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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