SARS-CoV-2 strain JPN/TY/WK-521 (59 (link)) was propagated in Vero-TMPRSS2 cells maintained in 2%-FBS/DMEM at 37°C in 5% CO2. Infectious titers of SARS-CoV-2 were determined in 50% tissue culture infectious dose (TCID50) assays using Vero-TMPRSS2 cells. Using VSV containing the green fluorescent protein (GFP) gene instead of the VSV G gene, VSV-SARS2 and VSV-EBOV were generated as described previously (60 (link), 61 (link)). Pseudotyped VSVs were pretreated with a neutralizing MAb to VSV G (VSV-G [N] 1-9) (33 (link)) to abolish the background infectivity of parental VSV. Pseudotyped VSVs were inoculated into each cell line cultured on 96-well plates, and infectious units (IU) were determined 20 h later by counting the number of GFP-expressing cells using IN Cell Analyzer 2500HS (GE Healthcare).
In cell analyzer 2500hs
Manufactured by GE Healthcare
Sourced in United States
The IN Cell Analyzer 2500HS is a high-speed, automated imaging system designed for cell-based assays. It captures and analyzes images of cells and cellular structures, providing quantitative data on various cellular parameters. The system utilizes advanced optics and image processing algorithms to enable high-throughput, multi-well plate screening applications.
Automatically generated - may contain errors
17 protocols using in cell analyzer 2500hs
1
Quantifying SARS-CoV-2 and Ebola Pseudotype Infectivity
2
SARS-CoV-2 spike protein pseudotyped VSIVs
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Using a replication-incompetent VSIV containing the green fluorescent protein (GFP) instead of the receptor-binding VSIV glycoprotein (G) gene (VSVΔG*-G), VSIVs pseudotyped with S proteins of SARS CoVs (VSVΔG*-SCoV and VSVΔG*-SCoV-2) were generated as described previously (41 (link), 42 (link)). Briefly, 24 h after transfection of HEK293T cells with pCAGGS expressing the S protein of SARS-CoV (Tor2 strain; GenBank accession number NC_004718.3 ) or SARS-CoV-2 (strain WHU01; GenBank accession number MN988668.1 ), the cells were incubated with VSVΔG*-G for 60 min at 37°C. After three washes with DMEM, the medium was replaced by DMEM with 10% FCS. After 24 h, the supernatants were harvested and stored at −80°C until use. Virus IUs in HEK293T cells were determined by counting the number of GFP-positive cells with an IN Cell Analyzer 2500HS (GE Healthcare). To produce VSIVs pseudotyped with the S proteins having the substitutions in RBD derived from the Alpha (N501Y), Beta (K417N, E484K, and N501Y), Gamma (K417T, E484K, and N501Y), and Delta (L452R and T478K) variants, the mutant S protein genes were constructed by site-directed mutagenesis with KOD One (Toyobo) based on the S protein gene of SARS-CoV-2 WHU01, which is an early isolate from Wuhan. Each mutation was confirmed by Sanger sequencing.
3
Quantifying SARS-CoV-2 Spike Protein Infectivity
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HEK293T cells, which are known to lack expression of endogenous ACE2 (45 (link)), were seeded in 96-well plates (1.0 × 104 cells per well) precoated with poly-l -lysine (Cultrex). After 24 h, the cells were transfected with 0.2 μg/well pCAGGS encoding FLAG-tagged ACE2 WT or SNV mutant proteins using TransIT-LT1 (Mirus). At 24 h posttransfection, these cells were infected with VSVΔG*-SCoV, VSVΔG*-SCoV-2, or VSVΔG*-G. Each virus was appropriately diluted to provide 300 to 400 IUs/well in HEK293T expressing WT ACE2. VSVΔG*-SCoV and VSVΔG*-SCoV-2 were treated with an anti-VSIV G monoclonal antibody [VSV-G(N)1-9] to abolish the background infectivity of parental VSVΔG*-G (46 (link)). After 24 h, the IUs were determined by counting the numbers of GFP-expressing cells by using an IN Cell Analyzer 2500HS (GE Healthcare). The relative infectivity was determined by setting the value of cells expressing ACE2 WT to 100%.
4
Quantifying SARS-CoV-2 Spike Protein Infectivity
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HEK293T cells, which are known to lack expression of endogenous ACE2 (45 (link)), were seeded in 96-well plates (1.0 × 104 cells per well) precoated with poly-l -lysine (Cultrex). After 24 h, the cells were transfected with 0.2 μg/well pCAGGS encoding FLAG-tagged ACE2 WT or SNV mutant proteins using TransIT-LT1 (Mirus). At 24 h posttransfection, these cells were infected with VSVΔG*-SCoV, VSVΔG*-SCoV-2, or VSVΔG*-G. Each virus was appropriately diluted to provide 300 to 400 IUs/well in HEK293T expressing WT ACE2. VSVΔG*-SCoV and VSVΔG*-SCoV-2 were treated with an anti-VSIV G monoclonal antibody [VSV-G(N)1-9] to abolish the background infectivity of parental VSVΔG*-G (46 (link)). After 24 h, the IUs were determined by counting the numbers of GFP-expressing cells by using an IN Cell Analyzer 2500HS (GE Healthcare). The relative infectivity was determined by setting the value of cells expressing ACE2 WT to 100%.
5
Quantifying Cell Proliferation with EdU
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The EdU incorporation assay was performed with Click-iT® Plus EdU Imaging Kits (Molecular Probes, Carlsbad, CA, USA). Briefly, cells were pulsed with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 4 h. Cells were fixed with 4% paraformaldehyde for 15 mins and subsequently washed twice with 3% BSA in PBS, followed by permeabilization with 0.5% Triton X-100 for 20 min at room temperature. After washing using 3% BSA, each slide was incubated with Click-iT Plus reaction cocktails including Alexa Flur 488 for 30 min at room temperature. For nuclear staining, cells were incubated with 5 μg/ml Hoechst 33342 solution for 30 min at room temperature. Numbers of EdU-positive and Hoechst-positive cells were analyzed using an IN Cell Analyzer 2500 HS (GE Healthcare).
6
SARS-CoV-2 spike protein pseudotyped VSIVs
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Using a replication-incompetent VSIV containing the green fluorescent protein (GFP) instead of the receptor-binding VSIV glycoprotein (G) gene (VSVΔG*-G), VSIVs pseudotyped with S proteins of SARS CoVs (VSVΔG*-SCoV and VSVΔG*-SCoV-2) were generated as described previously (41 (link), 42 (link)). Briefly, 24 h after transfection of HEK293T cells with pCAGGS expressing the S protein of SARS-CoV (Tor2 strain; GenBank accession number NC_004718.3 ) or SARS-CoV-2 (strain WHU01; GenBank accession number MN988668.1 ), the cells were incubated with VSVΔG*-G for 60 min at 37°C. After three washes with DMEM, the medium was replaced by DMEM with 10% FCS. After 24 h, the supernatants were harvested and stored at −80°C until use. Virus IUs in HEK293T cells were determined by counting the number of GFP-positive cells with an IN Cell Analyzer 2500HS (GE Healthcare). To produce VSIVs pseudotyped with the S proteins having the substitutions in RBD derived from the Alpha (N501Y), Beta (K417N, E484K, and N501Y), Gamma (K417T, E484K, and N501Y), and Delta (L452R and T478K) variants, the mutant S protein genes were constructed by site-directed mutagenesis with KOD One (Toyobo) based on the S protein gene of SARS-CoV-2 WHU01, which is an early isolate from Wuhan. Each mutation was confirmed by Sanger sequencing.
7
Whole-Slide Imaging of Tumor Microenvironment
8
Multiplexed Imaging of Tumor Microenvironment
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Images were acquired by the INCell Analyzer 2500HS (GE Healthcare) beginning with a whole slide image of the LN tissue at the 10X objective using DAPI and Cy3 fluorescent channels and was projected as a virtual H&E image for pathology review. Together with a serial H&E section, histopathological regions of interest were selected by a board-certified pathologist encompassing areas of tumor core, tumor-immune interface and lymphoid tissue. Individual fields of view (2040 x 2040 pixels, ~1mm2) were then selected from these histopathological regions at the 20X objective and used for all downstream multiplexed imaging (Supplemental Figure 1
9
Quantitative Analysis of Cell Migration
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HTR8/SVneo cell migration was determined by wound healing assays and performed in siC and siD7 as previously described [15 (link)]. To perform cell migration assay in stable shC and shD7 cells, they were seeded on twelve-well plates (1 × 105 cells/well) and cultured to reach a confluence of 70%. Then, cells were transfected or not with EV-GFP, p-Cx43-GFP, EV, D7.I, or D7.II plasmids for 48 h, as indicated. Confluent monolayers were wounded and assessed under microscopy at 0 and 8 h after. The results were expressed as the percentage of the wound closure calculated as the difference between the remaining area at 8 h and the initial one at 0 h recorded in seven fields of duplicate wells from three independent experiments.
Analysis of the migration of individual cells was conducted employing an IN Cell Analyzer 2500 HS (High content analysis, GE Healthcare Life Sciences) in a time-lapse mode (one picture every 15 min interval over a total time of 120 min). Individual cell trajectories were manually tracked using Image J/ Fiji software. Only cells at the front line of migration were examined, using the border of the cell as the reference mark for cell movement. Movies are presented in theS1 –S3 Movies.
Analysis of the migration of individual cells was conducted employing an IN Cell Analyzer 2500 HS (High content analysis, GE Healthcare Life Sciences) in a time-lapse mode (one picture every 15 min interval over a total time of 120 min). Individual cell trajectories were manually tracked using Image J/ Fiji software. Only cells at the front line of migration were examined, using the border of the cell as the reference mark for cell movement. Movies are presented in the
10
Quantifying SARS-CoV-2 and Ebola Pseudotype Infectivity
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SARS-CoV-2 strain JPN/TY/WK-521 (59 (link)) was propagated in Vero-TMPRSS2 cells maintained in 2%-FBS/DMEM at 37°C in 5% CO2. Infectious titers of SARS-CoV-2 were determined in 50% tissue culture infectious dose (TCID50) assays using Vero-TMPRSS2 cells. Using VSV containing the green fluorescent protein (GFP) gene instead of the VSV G gene, VSV-SARS2 and VSV-EBOV were generated as described previously (60 (link), 61 (link)). Pseudotyped VSVs were pretreated with a neutralizing MAb to VSV G (VSV-G [N] 1-9) (33 (link)) to abolish the background infectivity of parental VSV. Pseudotyped VSVs were inoculated into each cell line cultured on 96-well plates, and infectious units (IU) were determined 20 h later by counting the number of GFP-expressing cells using IN Cell Analyzer 2500HS (GE Healthcare).
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