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Black 96 well black bottom plates

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Black 96 well black bottom plates are a type of lab equipment used in various scientific applications. These plates feature 96 individual wells with black bottoms, which are designed to minimize background luminescence and optimize light detection for assays such as cell-based assays, biochemical assays, and luminescence-based measurements.

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Lab products found in correlation

Spectramax gemini em fluorometer, by Thermo Fisher Scientific (5 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) Elastin, by Thermo Fisher Scientific (1 mentions) Boc val pro arg amc, by Bachem (1 mentions) Gelatin, by Thermo Fisher Scientific (1 mentions)

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96-well plates (3030 citations) Black 96-well plate (168 citations) 96 wells (10 citations)

5 protocols using black 96 well black bottom plates

1

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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2

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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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

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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5

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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