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Spectramax gemini em fluorometer

Manufactured by Thermo Fisher Scientific
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The SpectraMAX Gemini EM is a fluorometer designed for quantitative and qualitative analysis of fluorescent samples. It measures fluorescence intensity and provides absorbance and luminescence capabilities. The instrument offers adjustable excitation and emission wavelengths to accommodate a wide range of fluorescent probes and dyes.

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Lab products found in correlation

Black 96 well black bottom plates, by Corning (5 mentions) Boc val pro arg amc, by Bachem (2 mentions) 96 well black bottom plates, by Corning (2 mentions) 5 fam lys leu leu asp ala ala pro lys qxl520 oh, by AnaSpec (2 mentions) Bicinchoninic acid (bca), by Bio-Rad (2 mentions) Boc val pro arg amc, by Teknova (2 mentions) Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions) Elastin, by Thermo Fisher Scientific (1 mentions) Gelatin, by Thermo Fisher Scientific (1 mentions) Enzchek elastase assay kit, by Thermo Fisher Scientific (1 mentions) 1 10 phenanthroline, by Merck Group (1 mentions) Dq collagen 4, by Thermo Fisher Scientific (1 mentions) Dq gelatin, by Thermo Fisher Scientific (1 mentions) Black 96 well plate, by Corning (1 mentions) Enzchek gelatinase collagenase assay kit, by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions) Aprotinin, by Merck Group (1 mentions) Dq elastin, by Thermo Fisher Scientific (1 mentions)

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SpectraMax Gemini (4 citations)

8 protocols using spectramax gemini em fluorometer

1

Quantifying Skin Trypsin Activity

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NHEK conditioned medium was added at 50 μl to 96-well black-bottom plates (Corning) followed by addition of 150 μl of the peptide Boc- Val-Pro-Arg-AMC (trypsin-like substrate; Bachem) at a final concentration of 200 μM in 1× digestion buffer [10 mM tris-HCl (pH7.8)] and incubated at 37°C for 24 hours. Relative fluorescence intensity (excitation, 354 nm; emission, 435 nm) was analyzed with a SpectraMax Gemini EM fluorometer (Thermo Fisher Scientific). For murine skin trypsin activity analysis, 0.5-cm2 full-thickness skin was bead-beat (2.0-mm zirconia beads, 2 × 30 s with 5 min after each) in 1 ml of 1 M acetic acid, followed by an overnight rotation at 4°C. Samples were centrifuged (10 min, 13,000 rpm, 4°C) and then added to a new microcentrifuge tube followed by protein concentration using a SpeedVac to remove all remaining acetic acid. Proteins were resuspended in molecular-grade water (500 μl) and rotated overnight at 4°C followed by another centrifugation. Clear protein lysates were added to a new tube, and bicinchoninic acid (Thermo Fisher Scientific) analysis was used to determine protein concentration. Last, 10 μg of total protein was added to a 96-well plate followed by analysis with the trypsin substrate as above.
Williams M.R., Costa S.K., Zaramela L.S., Khalil S., Todd D.A., Winter H.L., Sanford J.A., O’Neill A.M., Liggins M.C., Nakatsuji T., Cech N.B., Cheung A.L., Zengler K., Horswill A.R, & Gallo R.L. (2019). Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis. Science translational medicine, 11(490), eaat8329.
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2

Measuring Proteolytic Activities in NHEK Cells

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NHEK conditioned medium was added at 50µL to black 96 well black bottom plates (Corning, Corning, NY) followed by addition of 150µL of 5µg-mL BODIPY FL casein substrate, 2µg-mL of elastin (elastase-like substrate; ThermoFisher Scientific), or 4µg-mL gelatin (MMP substrate; ThermoFisher Scientific) according to manufacture’s instructions. Additionally, 200µM of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate; BACHEM, Bubendorf, Switzerland) was added to NHEK conditioned medium at 150µL in 1x digestion buffer (ThermoFisher Scientific). Relative fluorescent intensity was analyzed with a SpectraMAX Gemini EM fluorometer (ThermoFisher Scientific) at RT with readings every 2h for 24h. BODIPY FL casein plates were read at ex: 485nm and em: 530nm. elastin-like and MMP substrate plates were read at ex: 485nm and em: 515nm. Trypsin-like substrate plates were read at ex: 354nm and em: 435nm.
Williams M.R., Nakatsuji T., Sanford J.A., Vrbanac A.F, & Gallo R.L. (2016). Staphylococcus aureus induces increased serine protease activity in keratinocytes. The Journal of investigative dermatology, 137(2), 377-384.
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3

Quantifying S. epidermidis Protease Activity

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For the measurement of S. epidermidis protease activity, bacteria were grown for 24h in 3%TSB at 37°C before preparation of filtered-sterilized supernatant. The supernatant was tested for ecpA activity using a specific FRET substrate with the sequence (5-FAM)-Lys-Leu-Leu-Asp-Ala-Ala-Pro-Lys-(QXL520)-OH (AnaSpec, Fremont, CA) (Olson et al., 2014 (link)). 25 μl of S. epidermidis supernatant was added to black 96 well black bottom plates (Corning) followed by addition of 25 μL of 1x digestion buffer (10 mM Tris-HCl pH7.8, Teknova) containing the ecpA FRET substrate (1nM final). Relative fluorescent intensity (excitation: 485nm, emission: 538nm) was measured with a SpectraMAX Gemini EM fluorometer (Thermo Fisher Scientific) at t = 0 and after incubation at 37°C for 24h.
Williams M.R., Cau L., Wang Y., Kaul D., Sanford J.A., Zaramela L.S., Khalil S., Butcher A.M., Zengler K., Horswill A.R., Dupont C.L., Hovnanian A, & Gallo R.L. (2020). Interplay of Staphylococcal and Host Proteases Promotes Skin Barrier Disruption in Netherton Syndrome. Cell reports, 30(9), 2923-2933.e7.
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4

Protease Activity Assays for CoNS

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The protease activity assays were performed by using the EnzChek Elastase Assay Kit and EnzChek Gelatinase/Collagenase Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 20 μL of bacteria supernatant from various CoNS strains (see Table E2 in this article’s Online Repository at www.jacionline.org ) was incubated with 1 μg of DQ gelatin, DQ elastin, DQ collagen I, or DQ collagen IV (Thermo Fisher Scientific) in the supplied digestion buffer in 96-well black plates (Corning, NY) for 20 hours, in the presence or absence of various protease inhibitors: E64 (Sigma-Aldrich) at 5 μM; EDTA (Thermo Fisher Scientific) at 2 mM; 1,10-phenanthroline (Sigma-Aldrich) at 2 mM; or aprotinin (Sigma-Aldrich) at 0.5 mg/mL. Relative fluorescent intensity was analyzed with a SpectraMAX Gemini EM fluorometer (Thermo Fisher Scientific; excitation wavelength, 485 nm; emission excitation wavelength, 538 nm).
Cau L., Williams M.R., Butcher A.M., Nakatsuji T., Kavanaugh J.S., Cheng J.Y., Shafiq F., Higbee K., Hata T.R., Horswill A.R, & Gallo R.L. (2020). Staphylococcus epidermidis protease EcpA can be a deleterious component of the skin microbiome in atopic dermatitis. The Journal of allergy and clinical immunology, 147(3), 955-966.e16.
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5

Detecting Staphylococcus epidermidis agr Activity

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S epidermidis 12228 agr type I P3-GFPErmr (AH3408), S epidermidis 1457 agr type II P3-GFPErmr (AH273), and S epidermidis 8247 agr type IIIP3-GFP Ermr (AH3409) reporter strains were used to detect S epidermidis agr activity as previously described.32 (link) Strains were first cultured overnight (~16 hours) in the presence of 10 μg/mL of erythromycin (plasmid antibiotic selection). The bacteria were then inoculated at 107 CFUs/mL in 3% TSB (final volume 500 μL) along with CoNS sterile-filtered supernatant (≤10% vol/vol) or S hominis C5 synthetic AIP (≤100 nM) and shaken at 300 rpm in an incubator at 37°C. RNA was isolated after 12 hours of incubation, and the agr activity was measured after 24 hours. Bacteria were diluted 1:20 in PBS (final volume 200 μL) in 96-well black bottom plates (Corning), and GFP fluorescence was detected by using a SpectraMax Gemini EM fluorometer (Thermo Fisher Scientific; excitation wavelength, 485 nm; emission wavelength, 538 nm).
Cau L., Williams M.R., Butcher A.M., Nakatsuji T., Kavanaugh J.S., Cheng J.Y., Shafiq F., Higbee K., Hata T.R., Horswill A.R, & Gallo R.L. (2020). Staphylococcus epidermidis protease EcpA can be a deleterious component of the skin microbiome in atopic dermatitis. The Journal of allergy and clinical immunology, 147(3), 955-966.e16.
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6

Quantifying S. epidermidis Protease Activity

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For the measurement of S. epidermidis protease activity, bacteria were grown for 24h in 3%TSB at 37°C before preparation of filtered-sterilized supernatant. The supernatant was tested for ecpA activity using a specific FRET substrate with the sequence (5-FAM)-Lys-Leu-Leu-Asp-Ala-Ala-Pro-Lys-(QXL520)-OH (AnaSpec, Fremont, CA) (Olson et al., 2014 (link)). 25 μl of S. epidermidis supernatant was added to black 96 well black bottom plates (Corning) followed by addition of 25 μL of 1x digestion buffer (10 mM Tris-HCl pH7.8, Teknova) containing the ecpA FRET substrate (1nM final). Relative fluorescent intensity (excitation: 485nm, emission: 538nm) was measured with a SpectraMAX Gemini EM fluorometer (Thermo Fisher Scientific) at t = 0 and after incubation at 37°C for 24h.
Williams M.R., Cau L., Wang Y., Kaul D., Sanford J.A., Zaramela L.S., Khalil S., Butcher A.M., Zengler K., Horswill A.R., Dupont C.L., Hovnanian A, & Gallo R.L. (2020). Interplay of Staphylococcal and Host Proteases Promotes Skin Barrier Disruption in Netherton Syndrome. Cell reports, 30(9), 2923-2933.e7.
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7

Trypsin-like Substrate Fluorometric Assay

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NHEK conditioned medium was added at 50 μL to black 96 well black bottom plates (Corning) followed by addition of 150 μL of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate, BACHEM) at a final concentration of 200 μM in 1x digestion buffer (10 mM Tris-HCl pH7.8, Teknova) and incubated at 37°C for 24h. Relative fluorescent intensity (excitation: 354nm, emission: 435nm) was analyzed with a SpectraMAX Gemini EM fluorometer (Thermo Fisher Scientific). For murine skin trypsin activity analysis, 0.5cm2 full-thickness skin was bead beat (2.0mm zirconia beads, 2x 30sec with 5min on ice after each) in 1mL of 1M acetic acid followed by an overnight rotation at 4°C. Samples were centrifuged (10min, 13,000RPM, 4°C), added to a new microcentrifuge tube followed by protein concentration using a speedvac to remove all remaining acetic acid. Proteins were re-suspended in molecular grade water (500 μL) and rotated overnight at 4°C followed by another centrifugation. Clear protein lysates were added to a new tube, and BCA (Bio-rad) analysis used to determine protein concentration. Finally, 10 μg of total protein was added to a 96 well plate followed by analysis with the trypsin substrate as above.
Williams M.R., Cau L., Wang Y., Kaul D., Sanford J.A., Zaramela L.S., Khalil S., Butcher A.M., Zengler K., Horswill A.R., Dupont C.L., Hovnanian A, & Gallo R.L. (2020). Interplay of Staphylococcal and Host Proteases Promotes Skin Barrier Disruption in Netherton Syndrome. Cell reports, 30(9), 2923-2933.e7.
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8

Trypsin-like Substrate Fluorometric Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
NHEK conditioned medium was added at 50 μL to black 96 well black bottom plates (Corning) followed by addition of 150 μL of the peptide Boc-Val-Pro-Arg-AMC (trypsin-like substrate, BACHEM) at a final concentration of 200 μM in 1x digestion buffer (10 mM Tris-HCl pH7.8, Teknova) and incubated at 37°C for 24h. Relative fluorescent intensity (excitation: 354nm, emission: 435nm) was analyzed with a SpectraMAX Gemini EM fluorometer (Thermo Fisher Scientific). For murine skin trypsin activity analysis, 0.5cm2 full-thickness skin was bead beat (2.0mm zirconia beads, 2x 30sec with 5min on ice after each) in 1mL of 1M acetic acid followed by an overnight rotation at 4°C. Samples were centrifuged (10min, 13,000RPM, 4°C), added to a new microcentrifuge tube followed by protein concentration using a speedvac to remove all remaining acetic acid. Proteins were re-suspended in molecular grade water (500 μL) and rotated overnight at 4°C followed by another centrifugation. Clear protein lysates were added to a new tube, and BCA (Bio-rad) analysis used to determine protein concentration. Finally, 10 μg of total protein was added to a 96 well plate followed by analysis with the trypsin substrate as above.
Williams M.R., Cau L., Wang Y., Kaul D., Sanford J.A., Zaramela L.S., Khalil S., Butcher A.M., Zengler K., Horswill A.R., Dupont C.L., Hovnanian A, & Gallo R.L. (2020). Interplay of Staphylococcal and Host Proteases Promotes Skin Barrier Disruption in Netherton Syndrome. Cell reports, 30(9), 2923-2933.e7.
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