A modified ‘low pH’ lignocaine method to isolate human monocytes: A comparison with other separation procedures

Zanella, Alberto; Mantovani, Alberto; Mariani, Marcello; Silvani, Carlo; Peri, Giuseppe; Tedesco, F.

doi:10.1016/0022-1759(81)90191-5

Cited by 22 publications

(5 citation statements)

References 15 publications

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“…To detect OKMI-positive cells, the avidin-biotinperoxidase complex (ABC) technique (16) and monoclonal OKMl reagent (Ortho, Raritan, NJ) were used. To recover monocytes for addition to lymphocyte cultures, cells adherent to glass plates were detached by 20-minute incubation at room temperature and vigorous rinsing with 12 mM lidocaine in phosphate buffered saline (PBS), pH 7.4 (17). In some experiments, the isolated adherent cells were irradiated (3,000 rads for 5 minutes) prior to culture with Con A.…”

Section: Methodsmentioning

confidence: 99%

Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients

Whiteside

Worrall

Prince

et al. 1985

Arthritis & Rheumatism

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Dermal fibroblast cultures from patients with progressive systemic sclerosis (PSS) synthesize up to 5 times more glycosaminoglycan (GAG) than normal cultures. In an in vitro model of fibroblast-lymphocyte interactions, we show that the supernatants of activated mononuclear cells (MNC) modulate GAG synthesis, as measured by the incorporation of 3H-glucosamine into GAG following incubation of the confluent fibroblast monolayers with active supernatant preparations. GAG accumulation was selectively increased up to 18 times in normal dermal fibroblast cultures. Cell viability was dot affected, and 3H-thymidine uptake and cell numbers were depressed in cultures treated with the supernatants. In contrast to normal dermal fibroblast cultures, PSS fibroblasts responded to MNC supernatants by only a 1-2-fold increase in GAG. Supernatants of concanavaPresented in part at the 47th Annual Meeting of the American Rheumatism Association,

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Section: Methodsmentioning

confidence: 99%

Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients

Whiteside

Worrall

Prince

et al. 1985

Arthritis & Rheumatism

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“…Adherence of red cells to human monocytes. Normal human monocytes were isolated by the "low pH" lidocaine method, as described elsewhere [24]. Monolayers consisting of 2.5 x lo5 cells were prepared in the wells of Costar trays (Costar, Cambridge, MA); 30 x lo6 red cells in medium RPMI 1640 with 5.0 mM MgCI2 were added to each well.…”

Section: Methodsmentioning

confidence: 99%

Phosphofructokinase (PFK) deficiency due to a catalytically inactive mutant M‐type subunit

et al. 1982

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A case of M-type PFK deficiency due to the synthesis of a structurally abnormal and catalytically inactive M-subunit was reported. PFK activity was reduced (39% of normal) in red cells, normal in leukocytes and platelets, and absent in muscle. The red cell enzyme was not inhibited by antiserum to human muscle PFK and displayed normal biochemical properties (Km for ATP and fructose-6-phosphate, storage stability at +4 degrees C and -80 degrees C, optimum pH, electrophoretic pattern and molecular weight). The complete lack of PFK activity in muscle was confirmed on both histological preparations and muscle extracts. Double immunodiffusion analysis using an antinormal M-PFK serum revealed that the enzyme molecule was present and immunologically identical with normal, although it was catalytically inactive. The muscle abnormality was also confirmed by electromyography, ischemic exercise testing, histochemistry and electron microscopy. Moreover, PFK activity was investigated in myoblast cultures maintained up to 25 days, and it was found to be absent.

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“…The white cells were diluted with phosphatebuffered saline (PBS), and mononuclear cells were obtained by a centrifugation of 250g for 30 minutes over Ficoll (Lymphoprep) according to the method of Boyum. 25 After three washes with PBS, mononuclear cells were resuspended in RPMI 1640 containing 1% human serum at a density of 10 7 cells/mL. This cell suspension^ was placed in 24-well plates (0.5 mL/well) for biochemical experiments or in 25-cm 2 tissueculture flasks (10 mL/flask) for RNA studies.…”

Section: Cell Culturementioning

confidence: 99%

Essential differences in cholesteryl ester metabolism between human monocyte-derived and J774 macrophages. Evidence against the presence of hormone-sensitive lipase in human macrophages.

Contreras

Lasunción

1994

Arterioscler Thromb

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types, we looked for the presence of its mRNA in our macrophages by means of reverse transcription coupled to the polymerase chain reaction technique. Hormone-sensitive lipase mRNA was detected in J774 macrophages but not in human monocytes or in human monocyte-derived macrophages. These results demonstrated great differences in cholesteryl ester metabolism between macrophages of different origin. While hormone-sensitive lipase may be responsible for neutral cholesteryl ester hydrolytic activity in J774 macrophages, in human monocyte-derived macrophages it is not; thus, a different and as yet unidentified enzyme must be present. (Arterioscler Thromb. 1994; 14:443-452.) Key Words • human monocyte-derived macrophages • neutral cholesteryl ester hydrolase • hormone-sensitive lipase • cAMP • J774 macrophages at NORTH DAKOTA STATE UNIV on July 24, 2015 http://atvb.ahajournals.org/ Downloaded from at NORTH DAKOTA STATE UNIV on July 24, 2015 http://atvb.ahajournals.org/ Downloaded from

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A modified ‘low pH’ lignocaine method to isolate human monocytes: A comparison with other separation procedures

Cited by 22 publications

References 15 publications

Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients

Soluble mediators from mononuclear cells increase the synthesis of glycosaminoglycan by dermal fibroblast cultures derived from normal subjects and progressive systemic sclerosis patients

Phosphofructokinase (PFK) deficiency due to a catalytically inactive mutant M‐type subunit

Essential differences in cholesteryl ester metabolism between human monocyte-derived and J774 macrophages. Evidence against the presence of hormone-sensitive lipase in human macrophages.

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