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Infinite m200 spectrofluorimeter

Manufactured by Tecan

The Infinite M200 spectrofluorimeter is a laboratory instrument designed for fluorescence measurements. It provides accurate and reliable detection of fluorescent signals across a wide range of wavelengths. The core function of the Infinite M200 is to excite fluorescent samples and measure the emitted light, enabling quantitative analysis of fluorescent properties.

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5 protocols using infinite m200 spectrofluorimeter

1

Quantifying Plasma C3c Complement Binding

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We used serial plasma dilutions (10%, 5%, 2.5%, 1.25%, and 0.75% in BBS) and pooled plasma of mice belonging to the same experimental group, incubating the samples for 15 min on mannan- (10 µg/mL) or acBSA-coated (25 µg/mL) plates prepared according to a procedure reported previously.18 (link) Plates were washed and incubated for 1 h 30 min at RT with a polyclonal anti-human-C3c antibody (Dako, A0062) diluted to 2.4 µg/mL in wash buffer. After washing, plates were incubated with an alkaline phosphatase-labeled goat anti-rabbit IgG antibody (Sigma A-3812) diluted to 1 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of substrate solution (Sigma Fast p-Nitrophenyl Phosphate Tablets, Sigma). The absorbance at 405 nm was then measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).
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2

Quantification of MBL Isoforms in Plasma and Tissue

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MBL isoforms were analyzed after sample incubation on mannan-coated (10 µg/mL) plates prepared according to a procedure reported previously.18 (link) Plates were incubated for 1 h at 37 °C with EDTA-plasma diluted in barbital-buffered saline (BBS; 4 mm barbital, 145 mm NaCl, 2 mm CaCl2, 1 mm MgCl2 (pH 7.4)) to a final plasma concentration of 2.5% or with cortical homogenates (ipsilateral and contralateral to the lesion) diluted in BBS to a final concentration of 4 mg/mL total protein. Samples were then incubated for 1 h 30 min at room temperature (RT) with anti-MBL-A or anti-MBL-C antibodies (both 1 µg/mL, Hycult, # HM1035 and #HM1038, respectively). After washing, plates were incubated with an HRP-labeled goat anti-mouse IgG antibody (Santa Cruz, CA, USA), diluted to 0.4 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of TMB substrate solution (TMB Substrate Kit, code 34021, Thermo Scientific, MA, USA; diluted 1:1 in H2O2 solution). The reaction was terminated by adding 100 μL of 2 m H2SO4, and the absorbance at 450 nm was measured using an Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).
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3

EDTA-plasma Binding Assay for Anti-β2-GPI

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EDTA-plasma was diluted 1.25%, culture media was diluted 1.25, 2.5, 5 and 10% in BBS and incubated for 1 h 30′ at 37° C on plates coated overnight with a minibody obtained from the B2 clone of the anti-β2-GPI (MBB2, 0.06 µg/mL, see supplementary information). Next plate was incubated 1 h 30’ RT with anti-MBL-A, MBL-C or human MBL antibodies (1 µg/mL, Hycult). Absorption was read at OD 450 nm using the Infinite M200 spectrofluorimeter (Tecan, CH).
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4

Ex Vivo Complement Activation Post-TBI

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The LP activity was measured in mouse plasma samples obtained at 4 days after TBI or sham injury. For ex vivo LP assessment, EDTA-plasma samples (2.5% final plasma concentration) were incubated on mannan-coated ELISA plates for 15 min to initiate activation followed by detection of C3c deposition as described elsewhere [22 (link)]. Briefly, EDTA-plasma samples were thawed on ice and suspended in barbital buffered saline (BBS; 4 mM barbital, 145 mMNaCl, 2 mM CaCl2, 1mM MgCl2, pH 7.4), to a final plasma concentration of 2.5%. Plasma solutions were incubated on the coated plate at 37 °C for 15 min. The plate was washed and incubated for 1 h 30 min at RT with a polyclonal anti-C3c antibody (Dako, A0062) diluted 1:5000 in washing buffer. After washing, the plate was incubated with an alkaline-phosphatase labelled goat anti-rabbit IgG antibody (Sigma A-3812) diluted 1:5000 in washing buffer for 1 h 30 min at RT. Following washing, the assay was developed by adding 100 µL substrate solution (Sigma Fast p-Nitrophenyl Phosphate tablets, Sigma) and the absorption at 405 nm measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).
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5

Mannan-Induced Complement Activation Assay

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A functional lectin pathway activity assay was done using EDTA-plasma diluted 2.5% in barbital-buffered saline (BBS) incubated for 15′ at 37° C on 10 µg/mL mannan-coated plates.27 (link) C3 deposition was revealed using a polyclonal anti-human-C3c antibody (2.4 µg/mL, Dako) and an alkaline-phosphatase labeled goat anti-rabbit IgG (1 µg/mL, Sigma). Absorption at optical density (OD) 405 nm measured using Infinite M200 spectrofluorimeter (Tecan, CH).
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