iPSC-CMs were fixed for 15 min in 4% (v/v) paraformaldehyde (Thermo Fisher Scientific) in phosphate buffered saline (PBS) and permeabilized in 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific) for 15 min. Cells were blocked in 5% (w/v) bovine serum albumin (BSA) (Sigma) with 0.1% (v/v) Triton X-100 in PBS for 60 min. Primary antibodies were diluted in 5% (w/v) BSA and 0.1% (v/v) Triton X-100 in PBS and incubated overnight at 4°C. After treatment with primary antibodies, cells were washed three times in PBS for 15 min each. Secondary antibodies were diluted in 5% (w/v) BSA and 0.1% (v/v) Triton X-100 in PBS, and then incubated for 1 hr at room temperature. After treatment with the secondary antibody, cells were washed three times in PBS for 15 min each. Nuclei were stained using Hoechst 33342 (Thermo Fisher Scientific) (1:1000 dilution). Images were taken using a Cytation 5 microscope (BioTek Instruments) at 10× magnification (nine images per 384-well plate). A list of primary and secondary antibodies with the appropriate dilution is listed in Supplementary file 4 .
Cytation 5 microscope
Manufactured by Agilent Technologies
Sourced in United States
The Cytation 5 is a multi-mode microplate reader and imaging system from Agilent Technologies. It combines automated digital imaging and conventional microplate detection modes in a single, compact instrument. The Cytation 5 is capable of performing cell-based assays, fluorescence, luminescence, and absorbance detection.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Vectamount medium, by Vector Laboratories (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Gen5 imaging software, by Agilent Technologies (2 mentions)
Lipidspot lipid droplet stain, by Biotium (2 mentions)
Vectamount aq aqueous mounting medium, by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (1 mentions)
Tnf α elisa kit, by LSBio (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Formalin, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Uplfln 4 objective, by Agilent Technologies (1 mentions)
Oct media, by Thermo Fisher Scientific (1 mentions)
Cryotome cm1100, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab5622, by Abcam (1 mentions)
Ab28340, by Abcam (1 mentions)
21 protocols using cytation 5 microscope
1
Immunocytochemistry of iPSC-Derived Cardiomyocytes
2
Quantification of APA Expression in N2a Cells
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N2a cells were grown in 6-well plates at a density of 100 000 cells/well. Five days after infection, cells were fixed for 20 min with paraformaldehyde (PFA, 4%), then washed three times with PBS, permeabilized with Triton (0.1%) for 5 min and blocked for one hour with BSA (5%)/Tween (0.05%). Primary antibodies against APA were added overnight (1/500 dilution). Cells were then rinsed three times with PBS then incubated for one hour with secondary anti-Goat antibody (Interchim). Nuclei were stained with Dapi (1/20,000 dilution). Cells were then rinsed again with phosphate-buffered saline (PBS, 1X). Slices were mounted and cover-slipped with the Vectamount medium (Vector Laboratories) before imaging with the Biotek Cytation 5 microscope. Six fields per well were automatically acquired (brightfield and fluorescent images) using the acquisitions parameters (× 20 magnification, numeric aperture 0.45, and adapted excitation and emissions filters: DAPI (ex 377; em 447); GFP (ex 469; em 525); Txs Red (ex 586; em 647)). We used a macro in Image J software to quantify the red fluorescence intensity in GFP-positive cells.
3
Measuring Leukocidin-Induced Cell Death
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The leukocidin HlgAB was purified from S. aureus culture supernatants as described previously (10 (link)). Purified HlgAB was tested for LPS with the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript) and found to contain less than 0.006 EU/mL. HPMEC cells were grown and treated with isolated PMN for 6 hours or whole blood for 24 hours as above. The cells were incubated with 0.4 μM of HlgAB in serum-free EGM-2 media for 1 hour. Propidium iodide (PI, 10μg/ml) and Nucblue were then added to the media. The fraction of the total cells positive for PI was determined using the BioTek Cytation5 microscope and Gen5 software.
4
Trastuzumab Impact on BT474 Cell Viability and TNF-α Expression
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BT474, T-cells, and a co-culture of BT474 and T-cells were longitudinally imaged (N = 3 wells per group) to correlate changes in cancer cell viability with TNF-α expression. All experimental groups were either treated with 25 µg/mL trastuzumab or media (control) at t = 24 h. Treatment was removed at t = 48 h. On days 0 and 7, supernatant was collected for ELISA analysis. A TNF-α ELISA kit (LSBio. Catalog No. LS-F2557-1) was used to quantify TNF-α expression per sample per well. ELISA expression was quantified with a Cytation5 microscope (BioTek Instruments, Winooski, VT). Samples were normalized to expression on day 0. Samples were processed individually and averaged per timepoint. Because decreased cell viability was only observed in HER2 overexpressing cell lines, ELISA against TNF-α was only performed on BT474 cells due to their innate overexpression of HER2 protein.
5
Hypoxic Tumor Fate Mapping in NSG Mice
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Female 5- to 7-week-old NOD-SCID Gamma (NSG) mice were used according to protocols approved by the Johns Hopkins University Animal Care and Use Committee. Mice were anesthetized by the intraperitoneal (i.p.) injection of 100 mg/kg Ketamine, 16 mg/kg Xylazine, Vet One. MDA-MB-231 hypoxia fate mapping cells (2 × 106) were injected into the mammary fat pad (MFP) closest to the second nipple. Mice were i.p. injected with 1.25 mg of pimonidazole in saline (12.5 mg/mL) (Hypoxyprobe-1) 1 hr prior to sacrificing. Tumors were excised at various time points, formalin fixed (Sigma-Aldrich) for 1 hr, saturated in 30% sucrose (Sigma-Aldrich) at 4°C overnight, embedded in OCT media (Fisher Scientific), frozen in liquid nitrogen, sectioned via a cryotome CM1100 (Leica), and mounted onto Superfrost Plus microscope slides (Fisher Scientific). Tumor tissue sections were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1000 for 15 min, RT) and mounted with anti-fade solution. To assess the entire cross section of the tumor, slides were imaged with an Olympus (UPLFLN 4×) objective using Cytation 5 microscope (BioTek Instruments). Multiple image tiles were linearly stitched with Gen5 Software (BioTek Instruments).
6
Immunofluorescence Staining of Neuronal Markers
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C116 and HD MSN were fixed using 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (Corning, 21–040-CV) for 30 min. After three washes in PBS, cells were permeabilized and blocked for 1 h at RT using 0.1% Triton X-100 (Thermo Fisher Scientific, 28,313) and 4% donkey serum in PBS. Primary antibodies were added in the presence of blocking buffer ON at 4°C. Secondary antibodies (1:500) were added after three PBS washes in blocking buffer at RT for 1 h. The following primary antibodies were used for the immunofluorescence studies: rabbit anti-PPP1R1B/DARPP-32 (1:100; Santa Cruz Biotechnology, sc-11,365), rabbit anti-MAP2 (1:100; Millipore, AB5622) and rabbit anti-NES/nestin (1:100; Abcam, ab92391). The secondary antibodies were donkey anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen, A12379) or Alexa Fluor 647 (Invitrogen, A22287). Images were acquired using a Biotek Cytation 5 microscope and were prepared using Fiji software (ImageJ).
7
DPP4 Expression in N2a Cells
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Mouse neuroblastoma N2a cells (ATCC, CCL131) were grown in DMEM FCS (10%)-penicillin/streptomycin medium. Cells were plated in 6-well plates at a density of 100,000 cells/well. Five days after infection, cells were fixed for 20 min with paraformaldehyde (4%), washed three times with PBS, permeabilized for 5 min with Triton X-100 (0.1%), and blocked for 1 h with BSA (5%)/Tween (0.05%). The primary antibody against DPP4 (dilution of 1/500, Abcam ab28340) was added overnight, and then, cells were rinsed three times with PBS and incubated for 1 h with the secondary anti-goat antibody (Interchim, dilution of 1/500 + DAPI (1/20,000)). Cells were then rinsed again with PBS 1× and fixed with the VectaMount medium (Vector Laboratories) before Cytation imaging analysis with the Biotek Cytation 5 microscope, which automatically acquired six pictures per well with the same acquisition parameters (×20 magnification, numeric aperture 0.45, brightfield, at following excitation and emission wavelengths, respectively: DAPI (377; 447); GFP (469; 525); Texas Red (586; 647)). We used ImageJ software to quantify the fluorescence intensity Texas Red in GFP-positive cells.
8
Cellular Uptake of PBSe-GSK3787 Nanoparticles
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PBSe-GSK3787 particles were resuspended in cell media at concentrations of 0, 25, 50, 100, 150, 250, 500, 750, and 1000 μg/mL, and then sterilized under the UV light of the cell culture hood for 1 h. Then, 2 mL of suspension was added to the cell-containing wells of a 12 well plate and incubated for 48 h. Cells were imaged after 48 h of incubation with particles under bright field mode using a Biotek Cytation 5 microscope at 20× magnification.
9
Lung Tissue Histological Assessment
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Harvested lungs were fixed in 4% paraformaldehyde (Sigma‐Aldrich, USA) overnight before being embedded into paraffin and obtaining 5‐μm sections for haematoxylin and eosin (H&E) staining. H&E slides were imaged and analysed with Cytation‐5 microscope (Biotek, USA) and Metamorph Software (Molecular Devices, USA).
10
Immunohistochemical Analysis of EB Signaling
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Well-shaped EBs were selected and fixed in 4% paraformaldehyde-phosphate buffer salt solution fixative for more than 24 h. EBs were sedimented in sucrose solution, embedded in OCT, and sliced with a frozen section machine (CM1950, Leica, Wetzlar, Germany) at a thickness of approximately 12–15 μm. Subsequent frozen sections were fixed and antigenically repaired with EDTA antigen repair, endogenous peroxidase was blocked with 1% H2O2 for 10 min and 5% normal BSA to block nonspecific binding for 30 min. Sections were incubated with phosphate-Akt1 (1:200, servicebio, Wuhan, China) and phosphate-PI3K (1:100, abcam, Shanghai, China) overnight at 4 °C while PBS was added dropwise to each group of samples as a negative control. Subsequently, sections were incubated with goat anti-rabbit enzyme-labeled secondary antibody for 2 h at room temperature and diaminobenzidine for visualization. Finally, these sections were observed and analyzed using a Cytation 5 microscope (BioTek Instruments, Inc., headquartered in Winooski, VT, USA).
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