Direct toxicity assay on vaginal lactobacilli was performed using a colorimetric assay as described previously (39 (link)). TRF#8, TRF#30, TRF#36, and Lactobacillus crispatus LC223 were grown in the Lactobacillus MRS Broth (HiMedia™ Laboratories). Bacterial density was adjusted to an OD670 of 0.06, corresponding to a 0.5 McFarlands turbidity standard or ca. 108 CFU/ml. RfhSP-D was plated at the appropriate concentrations into a 96-well round bottom plates in a volume of 100 μl, and the diluted Lactobacillus spps were added in a volume of 100 μl. Commercially available penicillin-streptomycin solution (Gibco, Invitrogen) at a maximal test concentration of 1.25 U/ml and 1.25 μg/ml respectively) was used as a positive control for toxicity. Plates were incubated in an orbital shaker at 35°C under anaerobic conditions using AnaeroPack system (PML Microbiologicals, Wilsonville, OR) for 24 h. Bacterial growth was determined by measurement of the OD490 using a Victor2 counter with Wallac 2.01 software (PerkinElmer Life Sciences) (40 ).
Wallac 2
Manufactured by PerkinElmer
Sourced in United States, Germany
The Wallac 2.01 software is a data analysis tool designed for PerkinElmer's Wallac line of laboratory equipment. It provides users with the ability to analyze data generated from various experiments and assays performed on Wallac instruments.
Automatically generated - may contain errors
Lab products found in correlation
Victor2 counter, by PerkinElmer (2 mentions)
Bright glo luciferase assay system, by Promega (2 mentions)
Lactobacillus mrs broth, by HiMedia (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Ampliflu red, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
6 protocols using wallac 2
1
Lactobacilli Toxicity Assay Protocol
2
Evaluating rfhSP-D Impact on Cell Viability
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To assess the likely effect of rfhSP-D on cell viability, MTT assay was performed on Vk2/E6E7 and Ect1/E6E7 cell monolayers. Cells were seeded in a 96-well plate, grown up to confluence and then treated with a range of rfhSP-D concentrations at 37°C for 24 h. Culture supernatants were then collected for measuring immune mediators. Cells were treated with 1 × MTT containing KSFM and incubated overnight; 0.04 N acidified isopropanol was added to the cells to dissolve the formazan crystals. This color intensity, read at OD570, is directly proportional to the number of viable cells, as measured by a Victor2 counter with Wallac 2.01 software (PerkinElmer Life Sciences, USA) using a reference wavelength at 630 nm. The OD of untreated (medium alone) control cells was considered as 100%; percent viability of rfhSP-D treated cells was calculated as compared to untreated control.
3
NF-κB Activation Assay in Immortalized Epithelial Cells
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End1/E6E7 immortalized epithelial cells were transfected with pHTS–NF-κB firefly luciferase reporter vector (Biomyx Technology, USA) using a gene-juice transfection protocol (38 (link)). Cells were seeded in a 96-well plate, grown until confluent monolayers and treated with indicated concentrations of rfhSP-D for 24 h at 37°C. A synthetic analog of viral double-stranded RNA, Poly (I:C) (10 μg/ml) (InvivoGen, USA), a TLR3 agonist, and MALP-2 (25 nM), a TLR2/6 agonist, were used as positive controls. After incubation, the supernatant was removed, cells were lysed in GloLysis buffer, and activation of luciferase was determined using a Bright-Glo luciferase assay system (Promega, USA). Luminescence signal was quantified via a Victor2 1420 multi-label microplate counter with Wallac 2.01 software (PerkinElmer Life Sciences).
4
NF-kB Activation by Caco-2 and HT-29 Cells
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Caco-2 and HT-29 cells were transfected with pHTS-NF-kB firefly luciferase reporter vector (Biomyx Technology, San Diego, CA) and were cultured with LC2029 strain (Manifoldness of Infection (MOI) was 200 bacteria/cell), or in the presence of Slp2 (100 µg/ml), or MaLP-2 (50 nM) as a positive control. Supernatants were removed at the end of the treatment period, and the cells were lysed with GloLysis buffer. The luciferase activity was determined using the Bright-Glo Luciferase Assay System according to the manufacturer ' s protocol (Promega, Madison, WI). Luminescence was measured using a Victor 2 1420 multilabel microplate counter with Wallac 2.01 software (Perkin Elmer Life Sciences, Boston, MA).
5
Evaluating ECM-Mediated Drug Permeability
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The membranes of transwell inserts (24-well MWD format, 33 mm2 membrane area, Costar, Cölbe, Germany) were coated with 3 µg/mm2 of the respective ECM extract or protein by adding the protein suspension in 50 µL of buffer and letting the membranes air dry overnight. ECM was reconstituted by adding 150 µL of PBS to the upper chamber of the transwell and incubation for 1 h. Aliquot of 850 µL of 20 µg/mL doxorubicin in PBS were added to the lower compartment. The plate was read continuously for 6 h in a fluorescence plate reader (PerkinElmer, Wallac II; Ex: 530 nm, Em: 570 nm).
For LOX modification assays, 140 µg (10 µg/mm2) of matrigel were mixed with 10 µg purified recombinant hmLOX or hLOXL2 in 50 µL PBS (±500 µM BAPN), the suspension was applied to transwell inserts (96-well MWD format, 14 mm2 membrane area, Costar, Cölbe, Germany) and incubated for 6 h at 37 °C. Afterward, the suspension was dried overnight and subjected to the assay described above (100 µL PBS in upper chamber, 300 µL 20 µg/mL doxorubicin in PBS in lower chamber).
For LOX modification assays, 140 µg (10 µg/mm2) of matrigel were mixed with 10 µg purified recombinant hmLOX or hLOXL2 in 50 µL PBS (±500 µM BAPN), the suspension was applied to transwell inserts (96-well MWD format, 14 mm2 membrane area, Costar, Cölbe, Germany) and incubated for 6 h at 37 °C. Afterward, the suspension was dried overnight and subjected to the assay described above (100 µL PBS in upper chamber, 300 µL 20 µg/mL doxorubicin in PBS in lower chamber).
6
Fluorometric Assay for Lysyl Oxidase
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A modified form of the fluorometric microwell assay described by Fogelgren was used55 (link). In short: In a black 96-well microtiterplate was added to 120 μL of an freshly prepared assay solution consisting of 75 μL 2× PBS, 15 μL N-Acetyl-Resorufin (100 μM, Ampliflu-Red, Sigma-Aldrich), 15 μL 2,5-diaminopentane (100 mM, cadaverine a substrate of all five lysyl oxidases55 (link)56 (link)57 (link)) and 15 μL horseradish-peroxidase (5 U/mL, Sigma-Aldrich). For each sample the assay solution was prepared in two triplicate rows. To one triplicate 30 μL water, to the other 30 μL βAPN (3.5 mM in water) was added. Finally 50 μL of cell culture supernatant or protein containing lysate was added and the fluorescence of released resorufin was recorded continuously over 3 h in a fluorescence plate reader (PerkinElmer, Wallac II; Ex: 530 nm, Em: 570 nm). Slope of the fluorescence signal in the linear range was calculated for both βAPN-inhibited and non-inhibited samples; the difference was used as a measure of lysyl oxidase activity.
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