Archive for the ‘Cell’ Category - Part 3

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(12)

Washed sperm cells were also incubated in the absence of biotinylated carbohydrate conjugates with 100 |xl of streptavidin-FITC (negative control). The specimens were counterstained for cell viability using propidium iodide and only propidium iodide-negative cells (living cells) were imaged (single scans were made to freeze motile cells). Staining of the sperm was visualized using an […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(11)

Characterization of the SPM Binding to the Carbohydrate Probes by Saccharide Competition Assay Competition of SPM binding to the various prepared biotinylated carbohydrate conjugates was tested in polystyrene 96-well microtiter plates. The plates were coated with 0.5 |xg SPM protein/well diluted in coating buffer and plates were blocked as described for ELCBA. Various concentrations from […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(10)

A final concentration, 0.2 |xg/ml, of neutravidin-HRP was prepared in washing buffer and 50 ^l/well was added. After 1 h of incubation, free neu-travidin-HRP was removed and the plates were washed five times in washing buffer. Immobilized enzyme activity was quantified by adding 150 |xl/ well tetramethylbenzidine reagent (containing 100 |xg/ml tetramethylben-zidine, 1 mM H2O2 […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(9)

Enzyme-Linked Carbohydrate-Binding Assay Binding properties of bovine SPM to carbohydrates were analyzed by an enzyme-linked carbohydrate-binding assay (ELCBA). Polystyrene 96-well microtiter plates were coated with SPM samples (protein range from 1 ng to 8 |xg per well) diluted in coating buffer (containing 0.1 M Na2CO3, pH 9.6) and incubated overnight at 4°C. All further procedures […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(8)

Isolation of Glycosaminoglycans from Follicular Fluid GAGs were extracted from follicular fluid by alkaline borohydride. To 1 ml of follicular fluid, 3 ml borohydride buffer (containing 15 mM NaBH4 dissolved in 1 M NaOH) was added. After 1 h at 73°C, the mixture was neutralized by addition of 6 M HCl. Proteins were precipitated by […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(7)

Fucoidan was biotinylated with biotin-hydrazide after partial oxidation of the carbohydrate moieties according to the manufacturer’s instructions. Briefly, 10 mg fucoidan was dissolved in 200 |xl demineralized water and partially oxidized by the addition of 50 |xl of 100 mM NaIO4 (containing 100 mM CH3COONa, pH 5.5). After 15 min, the excess periodate was removed […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(6)

Folin-Ciocalteu phenol reagent (final concentration 0.1 M) was added for 30 min and color formation was measured in a Beckman DU-62 spectrophotometer (Beckman Instruments Nederland B.V., Mijdrecht, The Netherlands) at 750 nm. ii) Alkaline (EC 3.1.3.1) and acid (EC 3.1.3.2) phosphatases were assayed according to Soucek and Vary. iii) 5′-Nucleotidase (EC 3.1.3.5) activity was determined […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(5)

After 10 min, sperm cells were slowly extruded into a 50-ml polyethylene tube into which protease inhibitors were added. All further isolation steps were performed on ice or at 4°C. This procedure resulted in the exclusive removal of the apical sperm head membrane from sperm cells (cf., porcine sperm cells; data not shown). The cavitate […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(4)

Sperm was incubated in capacitating media (all T+BIC media) for 4 h in a humidified atmosphere at 38°C and 5% CO2, and sperm diluted in T—BIC medium was incubated in a humidified atmosphere at 38°C in air (containing 0.05% CO2) to prevent bicarbonate buffering of CO2. Sperm concentration in the media was 20 X 106 […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(3)

Semen was washed over a discontinuous Percoll density gradient. For this purpose, sperm cells were first layered on top of 35% and 70% isotonic Percoll layers diluted with saline medium as described by Harrison et al. and subsequently centrifuged at 350 X g for 10 min and 20 min at 700 X g at room […]

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