Bovine serum albumin (BSA) was chosen as a model protein for the evaluation of protein adsorption. First, 2 mg/mL BSA in PBS solution was prepared. PE membranes were incubated in protein solution for 24 h at 37 °C, and then washed with PBS and DI water five times to remove the weakly-bound proteins. Quantitatively, the bicinchoninic acid (BCA) protein assay kit was used to analyze the amount of adsorbed BSA where BSA was eluted from the sample by 1 wt % sodium dodecyl sulfate (SDS). The removed solution and BCA protein assay reagent were mixed in a ratio of 1:50. The absorbance was measured at wavelength 562 nm using a reader (iMark Absorbance Reader, BioRad, Hercules, CA, USA). A standard calibration curve was prepared based on the absorbance of a series of BSA concentrations.
Imark absorbance reader
Manufactured by Bio-Rad
Sourced in United States, Italy
The iMark Absorbance Reader is a microplate reader designed for measuring the absorbance of samples in microplates. It can be used for a variety of applications that require absorbance measurements, such as enzyme-linked immunosorbent assays (ELISAs), colorimetric assays, and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Mtt assay, by Bio-Rad (2 mentions)
Mtt solution, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay kit, by Promega (1 mentions)
Penicillin streptomycin pen strep solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
Sodium perborate buffer, by Merck Group (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Rapidout dna removal kit, by Thermo Fisher Scientific (1 mentions)
Revertaid rt reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Lucifer yellow lithium salt, by Thermo Fisher Scientific (1 mentions)
Cellstar 96 well culture plate, by Greiner (1 mentions)
13 protocols using imark absorbance reader
1
BSA Adsorption on PE Membranes
2
Cell Proliferation Assay via MTT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell proliferation/viability was determined by an MTT assay (Bio-Rad, Richmond, CA, USA). The assay was performed in 12-well plates, according to the manufacturer’s instructions (5 × 103/well). The absorbance was measured at 490 and 650 nm using an iMark™ Absorbance Reader from Bio-Rad (Richmond, CA, USA).
3
Cytotoxicity Assay for LDH Release
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lactate dehydrogenase (LDH) release was measured 24 h post-infection using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, UK) according to instructions. Non-infected monolayers were included for measurement of spontaneous LDH release and maximum LDH release was induced using the lysis reagent included in the kit. The absorbance was read at 492 nm using the iMark Absorbance Reader (Bio-Rad, Hercules, CA, USA).
4
MTT Assay for Cell Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
50 µl of MTT solution (Sigma-Aldrich, Milan, Italy) at 2.5 mg/mL concentration in HBSS (Hank’s buffered salt solution) pH 7.4 was added to cover each scaffold for 3 h. 22 Subsequently, MTT solution was removed from each well, and the substrates were washed with 200 µL of phosphate-buffered saline (PBS). After the removal of PBS, 100 µL of dimethyl sulfoxide (DMSO) was put in each well, and the absorbance was assayed at 570 nm by means of an enzyme-linked immunosorbent assay (ELISA) plate reader (Imark Absorbance Reader, Biorad, Segrate, Italy), with a reference wavelength of 690 nm. Cell viability was expressed as optical density (OD).
5
Biocompatibility Evaluation of Novel Biomaterials
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Biocompatibility (cytotoxicity) was evaluated using fibroblasts (normal human dermal fibroblasts, from juvenile foreskin from 2 to 5 passages, PromoCell, WVR, Italy).
Fibroblasts were grown in DMEM (Lonza, I), 10% v/v (FBS (EuroClone, Italy) and penicillin/streptomycin solution (pen/strep, 100 UI/100 μm/mL, Sigma Aldrich-Merck, Italy)
VHS (1.2 mg/mL), NF (0.1 mg/mL) and VHS-NF (VHS: 1.2 mg/mL and NF 0.1 mg/mL) were suspended/solubilized in cell culture medium and put in contact with the cells in suspension just after cell seeding in 96-well plate at seeding density of 35,000 cells/well. Fibroblasts were grown for 2 days. Fibroblast growth on tissue culture plastic was considered as standard growth (control).
At prefixed end point, cell growth was assessed by means of MTT test. Briefly, the medium was removed and 50 µL of MTT solution (Sigma Aldrich, Italy) at 2.5 mg/mL concentration in Hank’s Buffered Salt Solution pH 7.4 was added to cover each scaffold for 3 hrs. Subsequently, MTT solution was removed from each well, and the substrates were washed with 200 µL of PBS. After the removal of PBS, 100 µL of DMSO was put in each well, and the absorbance was assayed at 570 nm by means of an ELISA plate reader (Imark Absorbance Reader, Biorad, Italy), with a reference wavelength of 690 nm. Cell viability was expressed as optical density (OD).
Fibroblasts were grown in DMEM (Lonza, I), 10% v/v (FBS (EuroClone, Italy) and penicillin/streptomycin solution (pen/strep, 100 UI/100 μm/mL, Sigma Aldrich-Merck, Italy)
VHS (1.2 mg/mL), NF (0.1 mg/mL) and VHS-NF (VHS: 1.2 mg/mL and NF 0.1 mg/mL) were suspended/solubilized in cell culture medium and put in contact with the cells in suspension just after cell seeding in 96-well plate at seeding density of 35,000 cells/well. Fibroblasts were grown for 2 days. Fibroblast growth on tissue culture plastic was considered as standard growth (control).
At prefixed end point, cell growth was assessed by means of MTT test. Briefly, the medium was removed and 50 µL of MTT solution (Sigma Aldrich, Italy) at 2.5 mg/mL concentration in Hank’s Buffered Salt Solution pH 7.4 was added to cover each scaffold for 3 hrs. Subsequently, MTT solution was removed from each well, and the substrates were washed with 200 µL of PBS. After the removal of PBS, 100 µL of DMSO was put in each well, and the absorbance was assayed at 570 nm by means of an ELISA plate reader (Imark Absorbance Reader, Biorad, Italy), with a reference wavelength of 690 nm. Cell viability was expressed as optical density (OD).
6
ELISA Assay for BCG Antibody Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The assay was performed as previously described (4 (link), 5 (link)). Briefly, ELISA plates (Thermo Scientific) were coated with BCG antigen (1 μg/well) in coating buffer and incubated overnight before being washed with phosphate-buffered saline (PBS) and blocked with bovine serum albumin. Culture supernatant and positive/negative controls were run in duplicates; positive controls consisted of pooled sera from a well-characterized cohort of tuberculosis patients, and negative controls consisted of pooled sera from healthy controls from a setting of TB endemicity in Pakistan (35 (link)). PBS was used as a negative control. Plates were incubated for 2 h at 37°C. Secondary antibody (IgG) conjugated with horseradish peroxidase was added, and plates were incubated for 2 h with subsequent washing. Plates were finally developed using o-phenylenediamine tablets mixed in sodium perborate buffer (Sigma-Aldrich) and read at 490 nm using an ELISA reader (iMark absorbance reader; Bio-Rad, Hercules, CA). The absorbance was adjusted using “reagent blank” which contained BCG, conjugate, and substrate.
7
RNA Extraction and cDNA Synthesis from Yeast
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For extraction of RNA, cultures were grown in YEPD liquid medium until OD600 of 0.5–0.6 (iMark Absorbance Reader, BioRad, Hercules, CA, USA); then the cells were harvested and washed. Total yeast RNA was isolated using the GeneJET RNA Purification Kit (Thermo Scientific, Waltham, MA, USA, #K0731) and treated with DNase I (RapidOut DNA Removal Kit, Thermo Scientific, Waltham, MA, USA, #K2981) according to the manufacturer’s instructions. RNA concentration and quality were evaluated using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Purified RNA was reverse transcribed with RevertAid RT Reverse Transcription Kit (Thermo Scientific, Waltham, MA, USA, #K1691). cDNA generation was performed under the following conditions: 25 °C for 5 min, 42 °C for 60 min and termination at 70 °C for 5 min.
8
Transwell Permeability Assay ARPE-19 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Permeability assays were conducted with ARPE-19 cells cultured in Transwell inserts and co-cultured with conditioned media (Fig. 6 C). After being washed with PBS, the insert’s top and bottom chambers received 0.5 mL of 1 M Lucifer Yellow lithium salt (457.2 Da; Molecular Probes, CA, USA) and 1 mL PBS, respectively. After 2 h of incubation at 37 °C, 100 mL aliquots from the bottom chamber were transferred to a 96-well plate, and the absorbance was assessed using an iMark Absorbance Reader (Bio-Rad, CA, USA) at 450 nm.
9
Cell Viability Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability assays have been performed as previously described (36 (link)). Briefly, 3–5×103 cells were seeded into a 96-well plate (n = 4). 24 hours after drug application and over a period of 4 consecutive days, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT, 494.93 μmol/L) and phenazine methosulfate (4.2 μmol/L) were incubated for 4 hours at 37°C and measured by the iMark Absorbance Reader (Bio-Rad; absorbance: 450 nm; reference: 655 nm). EC50 values were calculated by GraphPad Prism v8.
10
Evaluating Cell Viability via MTT Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Proliferation assay (Sigma, St. Louis, MO, USA) 24 h after exposure to treatments. In brief, a total of 5000 cells/well were seeded into 12-well plate in a final volume of 1 ml. After treatments, 100 µl MTT solution (5 mg/ml in PBS) was added to the medium and cells were incubated at 37 °C for 4 h. Then, the supernatant was discarded and dimethyl sulfoxide was added to dissolve the formazan crystals. Treatments were carried out in triplicate. The optical density in each well was evaluated by measurement of absorbance at 490 and 650 nm using an iMark™ Absorbance Reader from Bio-Rad (Richmond, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!