Sheep brain tubulin was purified according to the method of Shelanski [29 (link)] by two cycles of assembly–disassembly and then dissolved in the assembly buffer containing 0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM of GTP (pH 6.6) to give a tubulin concentration of about 2–3 mg/mL. Tubulin assembly was monitored by fluorescence according to reported procedure [30 (link)] using DAPI as fluorescent molecule. Assays were realized on 96-well plates prepared with Biomek NKMC and Biomek 3000 from Beckman coulter (Villepinte, France) and read at 37 °C on Wallac Victor fluorimeter from Perkin–Elmer (Villebon-sur-Yvette, France). The IC50 value of each compound was determined as tubulin polymerization inhibition by 50% compared to the rate in the absence of compound. The IC50 values for all compounds were compared to the IC50 values of phenstatin and (-)-desoxypodophyllotoxin and were measured the same day under the same conditions.
Wallac victor fluorimeter
Manufactured by PerkinElmer
Sourced in France
The Wallac Victor fluorimeter is a versatile laboratory instrument designed for the detection and quantification of fluorescent signals. It utilizes advanced optical components and sensitive detectors to provide accurate and reliable measurements of fluorescent samples. The core function of the Wallac Victor fluorimeter is to enable precise fluorescence-based analyses and assays in a wide range of research and diagnostic applications.
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4 protocols using wallac victor fluorimeter
1
Tubulin Assembly Inhibition Assay
2
FITC-Dextran Absorption Assay in Mice
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The FITC-dextran assay was performed as previously described [21 (link)]. Briefly, mice were given 0.1 ml of 80 mg/mL FITC-dextran (Sigma; FD4) in PBS by enema 2h prior to sacrifice. Blood was collected by cardiac puncture and added to 3% acid-citrate dextrose; plasma was collected and fluorescence was measured using a Wallac Victor fluorimeter (Perkin-Elmer Life Sciences, Boston, MA).
3
Tubulin Assembly Inhibition Assay
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Sheep brain tubulin was purified according to the method of Shelanski [ 40 ] by two cycles of assembly-disassembly and then diluted in the assembly buffer containing 0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM GTP, pH 6.6 to a final concentration around 2-3 mg/mL. Tubulin assembly was monitored by fluorescence according to reported procedure [ 41 ] using DAPI as fluorescent molecule. Assays were realized on 96-well plates prepared with Biomek NKMC and Biomek 3000 from Beckman Coulter and read at 37°C on Wallac Victor fluorimeter from Perkin Elmer. The IC50 value of each compound was determined as the concentration required to decrease the maximum assembly rate of tubulin by 50% compared to the rate in the absence of compound. The IC50 values for all compounds were compared to the IC50 of isoCA-4 and isoerianin measured the same day under the same conditions.
4
Tubulin Assembly Inhibition Assay
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Sheep brain tubulin was purified according to the method of Shelanski [33] by two cycles of assembly-disassembly and then dissolved in the assembly buffer containing 0.1 M MES, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM GTP, pH 6.6
(the concentration of tubulin was about 2-3 mg/mL). Tubulin assembly was monitored by fluorescence according to reported procedure [34] using DAPI as fluorescent molecule. Assays were realized on 96-well plates prepared with Biomek NKMC and Biomek 3000 from Beckman Coulter and read at 37°C on Wallac Victor fluorimeter from Perkin
Elmer. The IC50 value of each compound was determined as the concentration which decreased the maximum assembly rate of tubulin by 50% compared to the rate in the absence of compound. The IC50 values for all compounds were compared to the IC50 of isoCA-4 and measured the same day under the same conditions.
(the concentration of tubulin was about 2-3 mg/mL). Tubulin assembly was monitored by fluorescence according to reported procedure [34] using DAPI as fluorescent molecule. Assays were realized on 96-well plates prepared with Biomek NKMC and Biomek 3000 from Beckman Coulter and read at 37°C on Wallac Victor fluorimeter from Perkin
Elmer. The IC50 value of each compound was determined as the concentration which decreased the maximum assembly rate of tubulin by 50% compared to the rate in the absence of compound. The IC50 values for all compounds were compared to the IC50 of isoCA-4 and measured the same day under the same conditions.
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