NRs + DOX, NRs + MTX and NRs-PMA were evaluated for cytotoxicity in vitro using SRB assay.10 Pre-seeded HepG2 and HT144 cells (>90% viability, 1.5 × 105 cells per ml: Falcon® 96-well, flat bottom, clear microplate) were exposed to 1 μg ml−1 of drug functionalized NRs for 24 hours at standard culture conditions. Untreated cells (NTC) and free drug controls (DOX and MTX = 0.01 μM) equivalent to total drug attached with NRs at 1 μg ml−1 dose were also included. An external MF (4 mT) was applied for 20 minutes using Helmholtz coil. After treatment, cells were fixed with TCA (50%) for 1 hour at 4 °C, washed with deionized water thrice and air dried. Cells were stained with 0.05% SRB dye for 30 minutes at room temperature, followed by washings (5×) with 1% acetic acid to remove excess. Air dried plates were then used to take photographs at 200× using Olympus CK2 light microscope with attached camera (Optika C-B10 digital camera). Photographs were analyzed using Optika Pro View software (Version: x86, 3.7.13977.20190224). Experiment was performed twice with triplicates of all samples. For better comparison, treatment groups without MF assistance, were also included in the study.
Pro view software
Manufactured by Optika
Sourced in Italy
Pro View is a software application designed for scientific analysis and data visualization. It provides users with tools to process and interpret experimental data obtained from various laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
C b10 digital camera, by Optika (3 mentions)
Ck2 light microscope, by Optika (2 mentions)
Im 3 led light microscope, by Optika (2 mentions)
Envision flex detection system, by Agilent Technologies (1 mentions)
Optical microscope, by Optika (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Optikam b5, by Optika (1 mentions)
10 protocols using pro view software
1
Cytotoxicity Assessment of Nanoparticle-Drug Conjugates
2
Quantifying Ki-67 and p53 Expression in Drug-Treated Cells
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Ki-67 and p53 protein expression was evaluated by ICC [73 (link)] (Dako EnVisionTM FLEX detection system). HepG2 and HT144 (>90% viability, 1.5 × 105 cells/mL) cells were cultured on sterile coverslips in 24-well plates. Cells were treated with IC50 doses of drug-loaded NPs for 24 h, followed by fixation with TCA and washing with deionized water. The cells were immersed in an antigen retrieval solution at 95 °C for 45 min. Endogenous peroxidases were blocked by adding a peroxidase blocker for 10 min. Ki-67 (clone MIB-1, working dilution 1:150) and p53 (clone DO-7, working dilution 1:50) mouse monoclonal antibodies were then added, and the cells were incubated at 4 °C overnight followed by the addition of HRP-conjugated secondary antibody (rabbit, polyclonal) for 30 min and DAB chromogen for 10 min to obtain the desired dark brown stain with washings in between. The cells were counter stained with hematoxylin, dehydrated, mounted, and observed under a light microscope (Nikon, MicroPhot-SA) at a 200× magnification with an attached camera (Optika C-B10 digital camera) and analyzed by using the Optika Pro View software (version x86, 3.7.13977.20190224). The percentage of antibody positive cells was calculated using the following formula:
3
Colony Formation Assay Protocol
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For the colony formation assay, cells were seeded in triplicate in 60 mm dishes (5 × 102 cells/dishes). Cells were incubated for 8 days at 37 °C and stained with violet crystal solution (0.05% violet crystal w/v, 1% formaldehyde, 1% PBS, 1% methanol, and deionized water) for 20 min at room temperature. The number of colonies was manually quantified. Demonstrative images of the colonies were obtained under an optical microscope (Optika Italy) and using Optika Proview software.
4
Cytotoxicity Screening of MFe2O4 NPs
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The cytotoxicity screening of colloidal drug-loaded MFe2O4 (M = Fe, Co, Ni, Zn) NPs was performed by using the SRB assay in vitro [30 (link)]. HepG2 and HT144 cells (>90% viability, 1.5 × 105 cells/mL) were seeded onto 96-well plates (Falcon® 96-well, flat-bottom, clear microplate) and treated with 5 µg/mL of drug-loaded NPs for 24 h at 37 °C, followed by fixation with 50% trichloroacetic acid (TCA) for 60 min at 4 °C. The plates were washed thrice with deionized water to remove TCA and then air dried. Afterwards, the SRB dye (0.05%) was added at room temperature for 30 min to stain the cells. The excess dye was washed with 1% acetic acid 4–5 times. After the plates were air dried, photographs were taken at a 200× magnification with an Olympus CK2 light microscope with an attached camera (Optika C-B10 digital camera) and analyzed using the Optika Pro View software (version x86, 3.7.13977.20190224). The experiment was performed twice with triplicates for all samples. The experimental controls included untreated cells, free doxorubicin, free methotrexate, and NPs-PMA.
5
Adipose Tissue Histomorphometric Analysis
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Epididymal white adipose tissue (eWAT), subcutaneous WAT (sWAT), perirenal (rWAT), mesenteric WAT (mWAT), and liver tissues were collected from the mice at sacrifice. The eWAT and liver were rinsed with ice-cold phosphate-buffered saline (PBS), fixed with a 10% formalin solution. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E). Images were captured on an IM-3 LED light microscope (Optika, Bergamo, Italy) equipped with the 8.3-megapixel digital camera (Optika) using the ProView software (Optika). Then, the adipocyte size was quantified using ImageJ software.
6
Quantifying Kidney Tissue Morphology
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Kidney tissues were harvested, removed the renal fascia, fixed with 10% neutral buffered formalin (Sigma), and then embedded in paraffin. The 4 μm thickness tissue sections were stained with periodic acid Schiff (PAS), and Masson’s trichrome (Masson) after deparaffinization and rehydration. Images were captured on an IM-3 LED light microscope (Optika, Bergamo, Italy) equipped with an 8.3-megapixel digital camera (Optika) using the ProView software (Optika). Then, the glomerulus size and renal fibrotic area were quantified using ImageJ software version 1.52a.
7
Liposome Visualization under Microscope
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On the glass slides, a small number of liposomes was put, and the coverslips were angled to prevent bubble forming. After placing the coverslip, the slide was observed under a microscope at 40X and the image was taken using Optika ProView software (Niu M,Tan Yn, Guan P, Hovgaard L, Lu Y, Qi J, 2014 ).
8
Histological Analysis of Testicular Artery
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For histological analysis, samples from different supratesticular zones of the supratesticular region of the testicular artery were washed with normal saline, and were fixed in Bouin's solution for 24 h. Fixed samples were then dehydrated through passing in an ethanol ascending series, and were cleaned in methyl benzoate and were embedded in paraffin wax. Sections of 5–8 µm thickness were cut and were stained with Harris hematoxylin and Eosin for general structure, Crossmon's trichrome for collagen and muscle fibers, and Wigert's Elastica for identification of elastic fibers21 . Sections were then examined using an OPTIKA B-293 microscope, and digital images were acquired by OPTIKA C-B10 camera and OPTIKA PRO View software.
9
Histological Analysis of Fish Samples
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After removal of the skin, samples with size of 1×1×0.5 сm were collected from each fish. The histological analysis was performed on 70 samples. The specimens were fixed immediately in 10% buffered formalin (pH 7.4), washed in running water, dehydrated in ascending alcohol series, cleared twice in xylene and embedded in paraffin. Cross sections of 5 µm were produced by means of YD-335A rotary microtome (China) and stained with haematoxylin/eosin. Histological evaluation was done on N-200 М biological microscope (Hangzhou Sumer Instrument Co., Ltd, China). Findings were photo-documented with digital camera Optikam B5 (Optika Srl, Ponteranica, Italy) and Optika Proview software (Optika Srl, Ponteranica, Italy).
10
Histological Evaluation of Intestinal Morphology
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After the fixation of the jejunum and ileum in a 10% neutral buffered formalin solution for histological evaluation, the organs were embedded in paraffin, cut to a thickness of 4-5 mm and stained with hematoxylin and eosin stains. Digital images were captured using a B-380 Optika microscope equipped with a C-P6 Pro camera and Optika ProView software (OPTIKA Microscopes, Ponteranica, Italy). Histological examinations were carried out according to the method performed by Shao et al. to determine VH, VW, VSA, CD, and VH to CD ratio. VSA was calculated for each villus by multiplying the VH by the VW by p [43 ].
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