Ace2+ and Ace2− HEK293 cells were seeded in 96 well plates at 5x10E4 cells per well in DMEM media supplemented with 10% FBS and incubated at 37 C. After 24 h, media was removed and replaced by equal amounts of all pseudovirus diluted in fresh DMEM media. After 18 hours, cell media was removed and cells were washed three times with HBS blocking buffer. Next, viral transduction was measured as neonGreen fluorescence using an IntelliCyt high throughput flow cytometer. Uninfected controls and the lentiviral particles expressing the RBD of the L strain were used to set the gates. All experiments were done in technical replicates and repeated in 3 different days, and statistical significances were calculated by unpaired t tests using the GraphPad Prism software version 9.0.
High throughput flow cytometer
Manufactured by Intellicyt
Sourced in United States
The Intellicyt high-throughput flow cytometer is a laboratory instrument designed for the analysis of cells and particles in a sample. It utilizes flow cytometry technology to rapidly measure and characterize multiple parameters of individual cells or particles as they pass through a laser beam. The core function of this equipment is to provide efficient and accurate data collection for a wide range of applications in biological research and drug discovery.
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Lab products found in correlation
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ique screener, by Intellicyt (1 mentions)
Crl 1435, by American Type Culture Collection (1 mentions)
Cellstripper, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
8 protocols using high throughput flow cytometer
1
Pseudovirus Transduction Assay in Ace2+ HEK293 Cells
2
Alanine Scanning of GP1,2ΔMLD Region
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Alanine scanning was performed by introducing alanyls (alanyls were changed to seryls) into GP1,2ΔMLD region 627–639 via site-directed mutagenesis of GP1,2ΔMLD-encoding plasmid. The plasmid clones were individually arrayed into 384-well plates and transfected into HEK 293T cells. Protein variants were cell surface-expressed for 22 h 21 (link). The indicated mAbs were incubated with the cells for 1 h before an Alexa Fluor 488-conjugated secondary antibody (Thermo Fischer Scientific) was added. Antibody binding was assessed by detection of cellular fluorescence with a high throughput flow cytometer (Intellicyt, Albuquerque, NM, USA). Background fluorescence was measured in vector-transfected control cells and mAb reactivity against the variants was calculated with respect to reactivity with GP1,2ΔMLD by subtracting the signal from mock-transfected controls and normalized to signals from wild-type GP1,2ΔMLD-transfected controls. Residues predicted to be involved in the epitope were identified when mAb and variant did not react, but when reactivity of other control mAbs was observed, which excludes glycoproteins variants that were misfolded or were expressed at low levels.
3
Binding of SWL-DNA trimers to PC-3 cells
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Binding of SWL-DNA trimers to PC-3 cells (purchased from ATCC (ATCC® CRL-1435™; Manassas, VA, USA) was assessed in duplicates on an Intellicyt high-throughput flow cytometer. The day before assaying, 1 × 104 PC-3 cells were seeded into a 96-well U-bottom plate. After serum starvation for 4 h, cells were washed with 1× PBS, treated with serial dilutions of Cy3 labeled DNA trimers conjugated to three, two, one or no peptide SWL (3xSWL-DNA trimer, 2xSWL-DNA trimer, 1xSWL-DNA trimer, 0xSWL-DNA trimer, respectively) for 30 min at room temperature. Subsequently cells were rinsed with 1× PBS, detached using 0.05% trypsin-EDTA and centrifuged at 1150× g for 5 min at room temperature. Cells were resuspended in 1× PBS/1% (w/v) bovine serum albumin (BSA) and analyzed by an iQue screener (Intellicyt, Albuquerque, NM, USA).
4
Neutralizing Antibody IC50 Estimation
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First, we estimated the IC50 of the four purified neutralizing antibodies on pseudotyped lentiviruses carrying the L strain RBD (fig. 3C ), and then used a concentration corresponding to 10 × the respective IC50s for the neutralization experiments. To estimate the IC50s of the neutralizing antibodies with pseudotyped lentivirus expressing the RBD of the L strain, 10-fold dilutions of the antibodies were incubated with L strain pseudotyped lentivirus for 1 hour at 37 C and subsequently added to ACE2+ HEK293 cells. NeonGreen fluorescence was analyzed using an IntelliCyt high throughput flow cytometer and the data from 4 different experiments were used to estimate the IC50 values using the Graphpad Prism version 9.0. For the neutralization experiments, equal amounts of the lentivirus pseudotyped with the L strain or the corresponding PV variants were pre-incubated with 10 × IC50 concentrations of the 4 antibodies for 1 hour at 37 C and subsequently added to ACE2+ cells. Technical duplicates and at least 3 biological replicates of each sample were performed. Statistical significance was calculated by unpaired t tests using the GraphPad Prism software version 9.0.
5
Shotgun Mutagenesis of EBOV Glycoprotein
6
Flow Cytometry Screening of E1E2 Mutants
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HEK-293T cells were plated in 384-well microplates and transfected with each E1E2 mutant construct (one mutant per well). Transfected cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). The cells were incubated with anti-E2 antibody for 1 h at room temperature, followed by a 30-min incubation with Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) in 10% normal goat serum (NGS). Cells were washed twice with PBS without calcium or magnesium (PBS−/−) and resuspended in Cellstripper (Cellgro, Manassas, VA) plus 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO). Cellular fluorescence was detected using an Intellicyt high-throughput flow cytometer (Intellicyt, Albuquerque, NM). Background fluorescence was determined by fluorescence measurement of vector-transfected control cells. MAb reactivities against each E1E2 mutant clone were calculated relative to wild-type reactivity after subtracting the signal from mock-transfected controls25 (link), 65 (link).
7
Shotgun Mutagenesis of EBOV Glycoprotein
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Shotgun mutagenesis was performed as previously described (Howell et al., 2016 (link)). Briefly, a library containing EBOV GP Mayinga with residues 33–676 mutated to alanine was generated and transfected into HEK293T cells in 384-well plates. Cells expressing the GP mutants were incubated with each primary antibody and then with Alexa-Fluor-488-conjugated secondary antibody. Binding was detected using an Intellicyt high-throughput flow cytometer (Intellicyt). Background was subtracted from control wells, and relative binding to each mutant was compared to wild-type controls. Mutants were identified as critical only if they lost binding to the test antibody but not to the internal controls (Davidson and Doranz, 2014 (link)).
8
Shotgun Mutagenesis of EBOV GP Binding
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