All FACS measurements were conducted using a FACS Celesta (BD Biosciences, Franklin Lakes, NJ, USA). PBMC were thawed rapidly at 37°C. After washing with cold PBS, cells were counted using a Neubauer counting chamber and diluted to desired concentrations. For FACS analysis, PBMC were blocked with human serum for 30 min at 4°C and incubated with a pre-titrated antibody cocktail including RTX-AF647 as well as AF750 (Thermo Fisher Scientific), for live-dead staining. For FACS analysis, in each sample at least 200,000 cells were regularly acquired. If not otherwise indicated, all antibodies were obtained from BioLegend (San Diego, CA, USA). The following antibodies and fluorophores were used: V450 anti-CD27 (clone: M-T271), V500 anti-IgD (clone: IA6-2), BV650 anti-CD3 (clone: OKT3), BV785 anti-CD45 (clone: HI30), FITC anti-CD38 (clone: HIT2; BD BioSciences), PE anti-IgM (clone: MHM-88), PerCP anti-CD4 (clone: L200), PE-Cy7 anti-CD19 (clone: HIB19), AF700 anti-CD8a (clone: HIT8a). PBMC were washed twice and measured using a FACS Celesta (BD BioSciences). Graphical analysis was performed using FlowJo version 10.6.1 (FlowJo, Ashland, OR, USA). Of note, in contour plots not all cells are depicted as single dots. For gating strategy see supplemental information (Figure S1
Facscelesta
Manufactured by BD
Sourced in United States, Germany, Canada
The BD FACSCelesta is a flow cytometry instrument designed for multicolor analysis of cells and particles. It features advanced optics and detectors to enable sensitive detection and enumeration of a variety of cell types and populations.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (22 mentions)
Facsdiva software, by BD (16 mentions)
Facsdiva, by BD (14 mentions)
Facsaria 2, by BD (13 mentions)
Facscalibur, by BD (11 mentions)
Propidium iodide, by Merck Group (9 mentions)
Perm wash buffer, by BD (9 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (9 mentions)
Cytofix cytoperm kit, by BD (8 mentions)
Lsrfortessa x 20, by BD (8 mentions)
Lsrfortessa, by BD (8 mentions)
Facscanto, by BD (6 mentions)
Ionomycin, by Merck Group (6 mentions)
Cell staining buffer, by BioLegend (6 mentions)
Golgistop, by BD (6 mentions)
Facsverse, by BD (5 mentions)
Mhcii, by BioLegend (5 mentions)
Human trustain fcx, by BioLegend (5 mentions)
Golgiplug, by BD (5 mentions)
Cytofix cytoperm, by BD (5 mentions)
605 protocols using facscelesta
1
Flow Cytometry Analysis of PBMCs
2
Quantifying Virus Infection Rates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells plated in 96-well plates were infected with rLCMV-mCherry or LCMV at an MOI of 1 for an adsorption period of 1 h at 37°C and subsequently cultured for 24 h. To analyze percent infected, cells were trypsinized and fixed in suspension with 4% PFA for 30 min. For infection with rLCMV-mCherry, analysis was done by flow cytometry on FACSCelesta (BD), where approximately 5,000 cells were recorded and gated based on SFC/SSC, FSC-H/FSC-A (singlets), and phycoerythrin-CF594 (mCherry) using FlowJo 10. For infection with LCMV, cells were permeabilized and stained for LCMV-N protein (primary, 113; secondary, Alexa Fluor 488) prior to flow gating for fluorescein isothiocyanate (FITC). Antibody details can be found in Table S3 .
For pseudotype infection assays, cells seeded in a 96-well plate were infected with various VSV-Arenavirus-GP pseudoviruses. At 24 hpi, cells were lifted using Tryple Select Enzyme (Gibco) and flowed on a FACSCelesta (BD) and used for FITC (enhanced green fluorescent protein) signal as previously described.
For pseudotype infection assays, cells seeded in a 96-well plate were infected with various VSV-Arenavirus-GP pseudoviruses. At 24 hpi, cells were lifted using Tryple Select Enzyme (Gibco) and flowed on a FACSCelesta (BD) and used for FITC (enhanced green fluorescent protein) signal as previously described.
3
Oxidative Stress and Mitochondrial Function Assay in PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral Blood Mononuclear Cells (2 × 105 cells) were incubated with 2 μM of DCFH‐DA dye (Sigma) at room temperature for 20 min. Following that, flow cytometry (FACS Celesta, BD Biosciences) was used to assess the level of DCF. Moreover, PBMCs (2 × 105 cells) were incubated with MitoSOX Red dye (Thermo Fisher), and co‐incubated with 50 nM of MitoTracker Green dye (Thermo Fisher) at 37°C for 30 min. Using flow cytometry (FACS Celesta, BD Biosciences), the fluorescence intensity of MitoSOX and MitoTracker Green was examined. The intensity of MitoSOX indicated mitochondrial ROS levels, then the intensity of MitoTracker green indicated mitochondrial mass levels, and finally, the ratio of MitoSOX/MitoTracker green was calculated.
15 (link)
15 (link)
4
SARS-CoV-2 Spike Subunit Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For SARS-CoV-2 spike subunit binding assay, cells were washed once with HBSS containing 2% FBS and incubated with 50 μg/mL S1-Fc, 200 μg/mL RBD-Fc (Sino biological, #40592-V08H) or NTD-Fc (Sino biological, #40591-V49H) for 30 min at 4°C, followed by washing two times with HBSS with 2% FBS. Cells were incubated with PE anti-human IgG for another 30 min at 4°C, washed two times, and fixed with 4% formaldehyde for 15 min. Cells were washed once, resuspended in HBSS with 2% FBS and analyzed by flow cytometry using FACSCelesta (BD Biosciences). To measure the surface expression of ACE2 and LRRC15, cells were washed with FACS buffer (1x PBS supplemented with 2% FBS and 1 mM EDTA) and stained with goat anti-ACE2 (R&D Systems, #AF933) at a 1:50 dilution or rabbit anti-LRRC15 (abcam, #ab150376) at a 1:100 dilution for 30 min at 4°C. Then the cells were washed two times and resuspended in FACS buffer containing the secondary antibodies at a 1:1000 dilution: AF647-labeled donkey anti-goat IgG (Invitrogen, #A32849) or AF488-labeled goat anti-rabbit IgG (Invitrogen, #A32731). After 30 min incubation at 4°C, the cells were washed two times, fixed with 4% formaldehyde for 15 min and washed and resuspended in FACS buffer before analyzing by flow cytometry using FACSCelesta (BD Biosciences) or Cytek Aurora spectral analyzer (Cytek Biosciences). Data was analyzed with Flowjo software.
5
Cell Cycle and Apoptosis Analysis of Dicentrine-Treated HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of the cells’ progression through the cell cycle phases, HeLa cells were treated with 10, 25 and 50 µM of Dicentrine for the indicated times. Both floating and adherent cells were harvested, washed twice with PBS 1× and finally fixed in 70% CH3CH2OH at 4 °C overnight. Next, cells were washed with PBS 1×, stained with 50 mg/mL of propidium iodide (PI; Invitrogen-eBioscience) in the presence of 75 kU/mL of RNase and analyzed using FACSCelesta (BD Biosciences, San Jose, CA, USA).
The evaluation of apoptosis was carried out following the Annexin V assay versus PI staining, as previously described [49 (link)]. Briefly, both floating and adherent cells were harvested and suspended in Annexin-binding buffer (final concentration of 1 × 106 cells/mL). Thus, cells were incubated with fluorescein isothiocyanate–Annexin V (Annexin-FITC) and PI for 15 min at RT away from the light and immediately analyzed by using FACSCelesta (BD Biosciences, San Jose, CA, USA). For each condition, 20,000 events were measured, setting 488 and 525 nm as the excitation and emission wavelengths, respectively. Data obtained were analyzed with the FACS Diva software (BD Biosciences).
The evaluation of apoptosis was carried out following the Annexin V assay versus PI staining, as previously described [49 (link)]. Briefly, both floating and adherent cells were harvested and suspended in Annexin-binding buffer (final concentration of 1 × 106 cells/mL). Thus, cells were incubated with fluorescein isothiocyanate–Annexin V (Annexin-FITC) and PI for 15 min at RT away from the light and immediately analyzed by using FACSCelesta (BD Biosciences, San Jose, CA, USA). For each condition, 20,000 events were measured, setting 488 and 525 nm as the excitation and emission wavelengths, respectively. Data obtained were analyzed with the FACS Diva software (BD Biosciences).
6
Cell Cycle and Apoptosis Analysis in Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell cycle assays, MDA-MB-231, MDA-MB-468, MCF7 and ZR75 cells were treated with vehicle or 20 μM Imipramine or 40 μM imipramine for 96 h and then fixed with 70% ethanol for at least 24 h before staining with propidium iodide, as previously described [30 ]. Cell cycle distribution was determined using a BD FACSCelesta™ or BD LSRFortessa™ X-20 instruments. For apoptosis assays, cells were harvested after 96 h treatment with Vehicle or 20 μM Imipramine or 40 μM imipramine and then stained with annexin V/propidium iodide mixture using the ApoAlert™ Annexin V-FITC Apoptosis Kit (Clontech, Cat # 630110). The percent of annexin V-propidium iodide-positive cells were determined using flow cytometry as described previously [30 ]. The samples were analyzed using a BD FACSCelesta™ or BD LSRFortessa™ X-20 instrument. Data were processed using BD FACSDiva™ or FlowJo v10.7.2 software and visualized using a GraphPad Prism program.
7
Cellular Internalization of PDGL Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the cellular internalization of different formulations, Cy5.5-NHS was conjugated to PDGL or PDGL@CAP. Pan 02 cells were seeded into 6-well plates (2 × 105 cells/well) and incubated overnight. PDGL-Cy5.5 or PDGL-Cy5.5@CAP (Cy5.5 at 3 μg/mL) were added in the presence of serum-free medium at pH 6.5 or 7.4. After incubation for 4 h, the cells were washed three times with cold PBS, and nanoparticle internalization was observed using confocal laser scanning microscopy (LSM 800, Zeiss, Germany). To quantify internalization, cells were trypsinized and redispersed in PBS for flow cytometry analysis (BD FACS Celesta, USA).
To explore the uptake mechanism, Pan 02 cells were pre-incubated for 2 h at pH 6.5 or 7.4 with chlorpromazine (10 μg/mL) to inhibit clathrin-mediated cellular uptake, nystatin (50 μg/mL) to inhibit caveola-mediated uptake, or amiloride (13.3 μg/mL) to inhibit macropinocytosis. Then the culture medium was removed, and cells were incubated with Cy5.5-labelled PDGL@CAP for another 2 h. Cells were washed three times with ice-cold PBS, trypsinized and analyzed by flow cytometry (BD FACS Celesta, USA).
To explore the uptake mechanism, Pan 02 cells were pre-incubated for 2 h at pH 6.5 or 7.4 with chlorpromazine (10 μg/mL) to inhibit clathrin-mediated cellular uptake, nystatin (50 μg/mL) to inhibit caveola-mediated uptake, or amiloride (13.3 μg/mL) to inhibit macropinocytosis. Then the culture medium was removed, and cells were incubated with Cy5.5-labelled PDGL@CAP for another 2 h. Cells were washed three times with ice-cold PBS, trypsinized and analyzed by flow cytometry (BD FACS Celesta, USA).
8
Analysis of Phosphorylated Histone H3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of pH3 was analysed using the BD FACSCelesta (BD Biosciences). CRC spheroids were made into single cells via trypsinisation as described in the section Generation of single cells and fixed in 70% ethanol. Samples were washed twice using PBS containing 0.1% Tween-20 and incubated with the primary antibody anti-phospho-Histone H3 (Ser10, mouse, Merck, 05–806, 1:500) in PBS containing 0.1% Tween-20 at RT for 2 h. Samples were subsequently washed twice and incubated with the secondary antibody goat anti-mouse IgG-Alexa Fluor 488 (Invitrogen, A11029, 1:600) overnight at 4 °C. Samples were washed twice using PBS containing 0.1% Tween-20 and DNA was stained with DAPI (1:500). Data were acquired using a BD FACSCelesta (BD Biosciences) and analysed with BD FACSDiva software version 8.0. Cell debris and doublets were excluded and 10.000 events were measured per experiment.
9
Cell Cycle and Apoptosis Analysis in Breast Cancer Cells
10
Quantification of PD-L1 and HLA-A,B,C Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PC3 and DU145 cells were stained with Brilliant Violet 421™ (BV421) mouse anti-human PD-L1 1:50 (CD274, Clone 29E.2A3, BioLegend, San Diego, CA, USA), PE mouse anti-human HLA-A,B,C 1:100 (Clone W6/32, BioLegend, San Diego, CA, USA), and eFluor™ 780 fixable viability dye 1:10000 (65,086,514, Invitrogen, Waltham, MA, USA). Staining was performed in PBS for 20 min at room temperature. Cells were analyzed using a BD FACSCelesta (Becton Dickinson, Franklin Lakes, New Jersey, USA) and FlowJo software (Tree Star, Ashland, Oregon, USA). The relative mean fluorescence intensity (MFI) was calculated as the MFI of each experimental sample/MFI of vehicle (DMSO) control.
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!