For the analysis of migration of non-adherent mononuclear cells a Boyden chamber assay was used. Briefly, cells were incubated with 2 µM Calcein-AM (ATT Bioquest, Sunnyvale, CA, USA) for 30 min to label the viable cells. After washing with PBS, cells (1 × 106 cells) were immediately applied to 8 µm cell culture inserts placed in a 24-well plate. The bottom part of the well contained the chemoattractant—either SCP-1 cell conditioned medium, MCP-1 or MCP-2. THP-1 cells migrated into the bottom part of the well within 6.5 h were quantified fluorometrically (λex = 485 nm and λem = 520 nm) with the omega microplate reader (BMG Labtech, Ortenberg, GER).
Omega microplate reader
Manufactured by BMG Labtech
Sourced in United Kingdom, Germany
The Omega microplate reader is a versatile and precise instrument designed for a wide range of applications in life science research and clinical diagnostics. It offers high-performance detection of absorbance, fluorescence, and luminescence in microplates, allowing users to accurately measure a variety of biological and chemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Clear f bottom medium binding, by Greiner (2 mentions)
5 and 6 chloromethyl 2 7 dichlorodihydrofluorescein diacetate acetyl ester cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Jc 10, by Enzo Life Sciences (2 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Raltegravir, by Santa Cruz Biotechnology (1 mentions)
Anti hiv 1 p24 antibody 39 5.4a, by Abcam (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Af1433, by R&D Systems (1 mentions)
T6066, by Merck Group (1 mentions)
P1379, by Merck Group (1 mentions)
Transwell membrane, by Corning (1 mentions)
15 protocols using omega microplate reader
1
Quantification of Cell Migration
2
Photorhabdus Growth Characterization
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The strains used in this study were the rifampicin sensitive parent strain of P. laumondii subsp. laumondii DJC (WT) (Zamora-Lagos et al., 2018 (link)), and a rifampicin resistant derivative of this strain, P. luminescens subsp. laumondii DJC (RifR) (Jones et al., 2010 (link)). Bacteria were grown in LB at 28°C unless otherwise stated. For growth on solid media, LB was supplemented with 1.5% agar and 0.1% sodium pyruvate and plates were incubated in the dark. When necessary, kanamycin was added to the media at a concentration of 25 μg/ml. To assess growth on solid media, overnight cultures of Photorhabdus were diluted to an OD600 of 0.05 and 5 μl were spotted on solid media. They were then incubated at the appropriate temperature for 48 h. To assess growth in liquid, the diluted cultures were incubated at the appropriate temperature for 24 h, shaking at 180 rpm. For continuous monitoring of growth in liquid, diluted cultures were aliquoted in flat bottomed 96-well plates and placed in a BMG Labtech OMEGA microplate reader at the appropriate temperature, with double orbital shaking at 300 rpm. Measurements of absorbance at 600 nm were taken in 30 min intervals.
3
ELISA Quantification of Secreted Proteins
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Cells were incubated in fresh half normal volume of media for 40 h prior to collection. Samples were cleared by centrifugation for 10 min at 4 °C at 8000g and then concentrated 2 times by Cryospin. Protein concentration was measured using DC Protein assay (BioRad) according to the manufacturer's protocol. Concentrated conditioned media were assessed using the respective Sandwich ELISA DuoSet Development System kits (R&D Systems, Abingdon, UK). The kits were tested and suitably modified to be transferred onto a 384-well plate platform with the use of an EpMotion 5075 Liquid Handling robot. All colorimetric measurements were performed at 450 nm with wavelength correction at 550 nm on an Omega microplate reader (BMG Labtech, Aylesbury, UK) applying a four-parameter logistic curve fit to the respective standard curves. Data were normalized to total protein concentrations.
4
Cytotoxicity Evaluation of Cefotaxime Complexes
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HepG2 cells were maintained in a DMEM medium supplemented with streptomycin, penicillin, and fetal bovine serum in 5% CO2 atmosphere at about 37 °C. The cell viability was assessed by SRB assay. Aliquots of about 100 μL of the cell suspension were seeded in well plates and immediately incubated in a complete medium for 24 h. The cells were then treated with another medium containing the drugs at various concentrations. After 3 days of exposure to the cefotaxime and cefotaxime metal complexes, the cells were fixed by replacing the medium with 10% TCA and incubated for 1 h at about 4 °C. The TCA solution was then removed, and the cells were washed 5 times with distilled H2O. Aliquots of the SRB solution were added and incubated at 37 °C in the dark for about 10 min. The well plates were then washed 3 times with acetic acid (1%) and air-dried. Next, 150 μL of TRIS were added to dissolve the protein-bound SRB stain. The absorbance was measured at 540 nm using a “BMGLABTECH®-FLUOstar” Omega microplate reader (Ortenberg, Germany) [21 (link)].
5
Kinetic Analysis of Chlorophyll Biosynthesis
6
Vpr Expression and Luciferase Assay
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The wild-type Vpr sequence was excised from pNL4.3 (ARP-3418, NIH AIDS Reagent Program) by digestion with PflMI and SalI restriction enzymes and ligated into pNL4-3.Luc.E−R− (ARP-114; NIH AIDS Reagent Program), digested in the same manner. Virus was prepared by transfection of 293T cells with these DNAs with a VSV-G expression construct. Before infection, the viral supernatants were checked for the presence and absence of Vpr by Western blot. Anti-Vpr polyclonal antibody (Proteintech, #51143-1-AP) and anti-HIV1 p24 antibody [39/5.4A] (Abcam, #ab9071) were used for staining. HeLa cells were treated with 10 µM Raltegravir (Santa Cruz Biotechnology, sc-364600) or the equivalent amount of dimethyl sulfoxide and infected with reporter viruses encoding Vpr or without Vpr. Luciferase activity was assessed 24 h after infection with the Luciferase Assay System (Promega, #E1501) according to the manufacturer’s instructions and measured on an Omega microplate reader (BMG Labtech).
7
Evaluating Trametinib and Ponatinib Cytotoxicity
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OPM2, Delta-47, MM.1S and H929 were cultured per ATCC recommendations at 37 °C, 5% CO2. Cells were cultured in RPMI-1640 media (Hyclone, cat# SH30027FS) supplemented with 10% FBS (Sigma, cat# F2442-500ML). H929 cells were also cultured in 1X 2-Mercaptoethanol (GIBCO, cat# 21–985-023). Cells were freshly passaged 24 h prior to drug testing. 100 µl of 0.3 × 106/ml cells were plated per well in 96-well plates with pre-added trametinib or ponatinib. 48 h later, cell viability was evaluated using CellTiter-Glo assay (Cat No. G7573, Promega). Chemiluminescence was measured using an OMEGA microplate reader (BMG Labtech).
8
Enzyme-Linked Immunosorbent Assay Protocol
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Phosphate buffered saline (PBS) (Sigma P4417-199TAB), Tris (50 mM, Sigma T6066) buffered saline (150 mM, Fisher Scientific S/3161/65) with Tween-20 (0.01%, Sigma P1379) (TBST) were prepared and used for the study. Microtiter ELISA plates were procured from Costar 9018, Thermo Fisher Scientific whereas the plate reader and RDS-2500 reader were purchased from FLUOstar Omega Microplate Reader (BMG Labtech) and (DETEKT, USA) respectively. Anti-species alkaline phosphatase (A5187 and SAB3700286) was procured from Sigma, USA. The substrate pNPP was procured from BioPanda, USA. The paired antibodies used in the study are summarized in Table 5 .
Paired antibody and recombinant protein standard used for the study.
| Antibody | Recombinant protein standard | |
|---|---|---|
| UMOD | MAB5144; AF5144 (R&D Systems, USA) | H00007369-Q01 (Novus Biologicals, USA) |
| OPN | MAB1433; MAB14332R; AF1433 (R&D Systems, USA) | 1433-OP (R&D Systems, USA) |
| IL-9 | MAB2091; AF209; MAB209 (R&D Systems, USA) | 209-ILB010 (R&D Systems, USA) |
9
Luminescent Cell-Based sPLA2 Assay
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For the bioassay, 2,500 cells per well in 100 μl DMEM medium with supplements were seeded in a sterile white 96-well plate. For the free luciferin assay the cells were incubated for 24 hours before use, whereas for testing the liposomes and to allow for accumulation of sufficient sPLA2 enzyme the cells were incubated for 48–72 hours before the test. The experiments were performed in duplicates or more, and the luminescence signal from the luciferase-luciferin reaction was recorded in a FLUOstar Omega Microplate Reader (BMG LAB- TECH) thermostated at 37°C. After the equilibration of the solutions and the microplate seeded with the living MCF-7 cells at 37°C for few minutes, the bioassay was started by the automated injection via machine pumps of free luciferin or luciferin-loaded liposomes (intact or lysed) to the cells. Luciferin and Triton X-100 solutions were prepared in 120 mM potassium sulfate buffer (pH 6.0).
10
Transwell Cell Migration Assay
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Cells were disaggregated as single cell suspension using trypsin, washed twice with PBS, and stained with PKH26 labeling solution according to manufacturer’s protocol (Sigma). After washes, 5 × 105 cells were carefully resuspended in 100 µl of DMEM 0.1% BSA and seeded over a Transwell membrane (Corning) previously placed in a 24‐well plate. Cells were allowed to settle for 10 min on the membrane; then, 400 µl of DMEM 10%FBS was added in the bottom of each well. The PKH26 fluorescence signal was acquired using Omega Microplate Reader (BMG Labtech) at the top and at the bottom of each well at different time points, and the fraction of migrated cells was calculated as following:
After the acquisition of the fluorescent signal, the Transwell membranes were removed from the 24‐well plates and the cells, able to pass through the membranes, were allowed to attach for 8 h to capture representative images of the cells that efficiently migrated over the 2‐h period.
After the acquisition of the fluorescent signal, the Transwell membranes were removed from the 24‐well plates and the cells, able to pass through the membranes, were allowed to attach for 8 h to capture representative images of the cells that efficiently migrated over the 2‐h period.
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