For doxorubicin-induced premature senescent cells, cells were resuspended at 2.5 × 103 cells per well to a 96-well black wall/clear bottom plate (Corning) with the culture medium containing 1/500 volume of Incucyte Nuclight Rapid Red Dye (Sartorius). The cells were incubated and stained in Incucyte SX5 (Sartorius) placed in a 37 °C/5% CO2 incubator for 6 h, then 5 photomicrographs per well were acquired every 3 h for following 48 h. Nuclight-positive nuclei per image were counted by Incucyte software (version 2021A).
Black wall clear bottom 96 well plate
Manufactured by Corning
The Black wall/clear bottom 96-well plate is a laboratory equipment product designed for use in various experimental procedures. The product features a black opaque wall construction with a clear bottom, allowing for improved light transmission and optical clarity during experiments. This plate is commonly used in applications that require well-plate based analysis or detection methods.
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Lab products found in correlation
Steadyglo reagent, by Promega (2 mentions)
Topcounter, by PerkinElmer (2 mentions)
Fluostar omega, by BMG Labtech (2 mentions)
P3566, by Thermo Fisher Scientific (2 mentions)
Incucyte sx5, by Sartorius (1 mentions)
Incucyte nuclight rapid red dye, by Sartorius (1 mentions)
Synergy 2 multi mode microplate reader, by Agilent Technologies (1 mentions)
Spectramax m2 microplate reader, by Molecular Devices (1 mentions)
Oxiselect intracellular ros assay kit green fluorescence, by Cell Biolabs (1 mentions)
Ab184154, by Abcam (1 mentions)
Amplex red assay, by Thermo Fisher Scientific (1 mentions)
Il 1β elisa kit, by Thermo Fisher Scientific (1 mentions)
Realtime glo annexin 5 apoptosis assay, by Promega (1 mentions)
Spectramax5e, by Molecular Devices (1 mentions)
Qbt fatty acid uptake kit, by Molecular Devices (1 mentions)
12 protocols using black wall clear bottom 96 well plate
1
Doxorubicin-induced senescence cell assay
2
Bioluminescent Monitoring of S. aureus Promoter Activity
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Overnight cultures of the S. aureus promoter-lux reporter strains were diluted to an OD600 ≈ 0.2 in fresh TSB supplemented with or without test compounds, as indicated. Then, 100 μl aliquot of the sample was distributed to a 96-well black-wall clear-bottom plate (Costar, Corning Incorporated), and 50 μl saxoline was added to each well to prevent evaporation. Promoter activities were measured using a Synergy 2 Multi-Mode Microplate Reader (Biotek) at different time points of bacterial growth. A similar procedure was used to measure the effects of tunicamycin on the expression of psmα-lux in the presence of exogenous addition of AIP, except that the bioluminescence activity and the OD600 were only monitored at 2 h. For the maintenance of plasmid pYJ335 or its derivatives, 120 μg/ml erythromycin was used, where appropriate.
3
Cellular Assays for ROS and Inflammation
4
CGRP Modulates eNOS Phosphorylation
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HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 105 cells/well) and maintained at 37 °C in 5% CO2 and 95% O2 for 24 h. HSCs and IMS32 cells were exposed to CGRP (1 and 10 μM, respectively) or its vehicle (phosphate-buffered saline, PBS) for 5, 10, 15, 30 and 60 min, at 37 °C, then washed with DMEM pH 2.5 and fixed in 4% paraformaldehyde for 30 min. Cells were then washed with TBST (0.05%) and blocked with donkey serum (5%) for 4 h at room temperature and incubated overnight 4 °C with eNOSpS1177 (#ab184154, rabbit polyclonal, 1:100, Abcam, Lot: GR3257047-9). Cells were then washed and incubated with donkey anti-rabbit IgG conjugated with horseradish peroxidase (HRPO, 1:2000, Bethyl Laboratories Inc.) for 2 h at room temperature. Cells were then washed and stained using SIGMAFAST OPD for 30 min protected from light. After the incubation period, the absorbance was measured at 450 nm. Change in NOS3 phosphorylation was calculated as a percentage of the signal in vehicle-treated cells.
5
H2O2 Detection in HSCs and IMS32 Cells
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H2O2 was determined by using the Amplex Red assay (#A12222, Thermo Fisher, Invitrogen). HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 105 cells/well) and maintained in 5% CO2 and 95% O2 (24 h, 37 °C). The cultured medium was replaced with Krebs-Ringer phosphate (KRP, composition in mM: 2 CaCl2; 5.4 KCl; 0.4 MgSO4; 135 NaCl; 10 D-glucose; 10 HEPES [pH 7.4]) added with olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM), A967079 (50 μM), PBN (50 μM) or vehicle (0.1% DMSO in KRP) for 20 min at room temperature. HSCs or IMS32 cells were then stimulated with CGRP (1 and 10 μM, respectively) or its vehicle (KRP) added with Amplex red (50 μM) and horseradish peroxidase (HRP, 1 U/ml) and maintained for 40 min at room temperature protected from light. Some experiments were performed in Ca2+-free KRP containing EDTA (1 mM). Signal was detected 60 min after exposure to the stimuli. H2O2 release was calculated using H2O2 standards and expressed as nmol/1.
6
Measuring Firefly Luciferase Activity
7
Kinetics of Cell Death and Apoptosis
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To measure live kinetics of cell death, 60,000 cells were seeded per well of a black-wall, clear-bottom 96-well plate (Corning) for differentiation with PMA, treated and infected as described above. Medium was replaced with phenol-red-free RPMI supplemented with 5 μg/mL propidium iodide (P3566, Invitrogen). The plate was sealed with a clear, adhesive optical plate seal (Applied Biosystems) and placed in a plate reader (Fluostar Omega, BMG Labtech) pre-heated to 37°C. PI fluorescence was recorded with top optics every 15 min for times as indicated.
Apoptosis kinetics were analyzed using the RealTime-Glo Annexin V Apoptosis Assay (JA1001, Promega) according to the manufacturer’s instruction. Simultaneously with infection, detection reagent was added. Luminescence was measured using a Fluostar Omega plate reader (BMG Labtech). No-cell, medium-only controls were used for background correction.
For IL-1β ELISA, the cell culture supernatant was harvested, cleared by centrifugation at 2000 x g for 5 minutes and diluted in the buffer provided with the ELISA kit. ELISA was performed according to the manufacturer’s instruction. IL-1β ELISA kit was from Invitrogen (#88-7261, detection range 2 - 150 pg/mL).
Apoptosis kinetics were analyzed using the RealTime-Glo Annexin V Apoptosis Assay (JA1001, Promega) according to the manufacturer’s instruction. Simultaneously with infection, detection reagent was added. Luminescence was measured using a Fluostar Omega plate reader (BMG Labtech). No-cell, medium-only controls were used for background correction.
For IL-1β ELISA, the cell culture supernatant was harvested, cleared by centrifugation at 2000 x g for 5 minutes and diluted in the buffer provided with the ELISA kit. ELISA was performed according to the manufacturer’s instruction. IL-1β ELISA kit was from Invitrogen (#88-7261, detection range 2 - 150 pg/mL).
8
Live Kinetics of Necrotic Cell Death
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To measure live kinetics of necrotic cell death, 0.06 × 106 cells were seeded per well of a black‐wall, clear‐bottom 96‐well plate (Corning) and differentiated with PMA, pre‐treated, and infected as described above. Just before the start of the assay, medium was replaced with RPMI without phenol red and supplemented with 5 μg/ml propidium iodide (PI, P3566, Invitrogen). Then, the plate was sealed with a clear, adhesive optical plate seal (Applied Biosystems) and placed in a plate reader (Fluostar Omega, BMG Labtech) pre‐heated to 37°C. PI fluorescence was recorded with top optics every 15 min for times as indicated.
9
Measuring Firefly Luciferase Activity
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293TLR3/IFNβLuc cells were seeded in black wall/clear bottom 96-well plate (Corning Inc.). The firefly luciferase activities, under different experimental conditions, were measured by adding equal volume of Steady-Glo reagent (Promega), followed by luminometry in a TopCounter (Perkin Elmer).
10
ROS Detection in Mtb Strains
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ROS detection was slightly modified from previous protocols (Howell Wescott et al., 2017; Kalyanaraman et al., 2012; Miller et al., 2011) as following. Mtb WT and mutant strains were grown in the presence of Z0933/Z0930 (1/43 MIC, 1/23 MIC, 13 MIC and 43 MICs) to mid-log-phase and OD 600 adjusted to 0.3-0.5. Samples were taken immediately before addition of drug (time zero) and then up to 3 hours. DMSO was used as a vehicle control. Bacterial cultures were harvested by centrifugation and re-suspended in 7H9 without OADC or phosphate buffer saline (PBS) containing 40 mM DCFDA (Abcam) or 10 mM HPF (ApexBio). Samples were incubated in the dark at 37 C for 30 min, harvested, washed, and resuspended in 7H9/PBS prior to measurement. A triplicate of 100 mL of each sample was loaded into a black-wall, clear-bottom 96-well plate (Corning). Fluorescence (DCFDA excitation 485/ emission 535, and HPF excitation 490/emission 520) was measured using a Mithras 2 LB 943 (Berthold Tech). Fold increase intensity was calculated by dividing the experimental treated sample with the vehicle control. Each experiment was performed three times. For the hydroxyl radical quenching experiments, 20 mM thiourea was added 30 min before, during, and after all the washing steps.
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