Flow cytometry was used to assess expression of alpha‐smooth muscle actin in the fibrosis model. 0.25 × 106 corneal fibroblasts were seeded into 6‐well plates in triplicates in DMEM/F‐12 10% FBS medium one day before treatment. Fibrosis was induced as described in the earlier section. Cells were harvested with 0.05% trypsin‐EDTA 2 days and 4 days after treatment, and washed two times with PBS. 1 × 106 cells/100 µL was aliquoted into FACS tubes (VWR #734‐0442). 0.5 mL of cold Flow Cytometry Fixation Buffer (R&D, #FC004) was added to each tube. Tubes were vortexed and incubated for 10 min at room temperature. Next, cells were washed two times with PBS and the pellet was resuspended in 200 µL of Flow Cytometry Permeabilization/Wash Buffer I (R&D, #FC005). 10 µL of human alpha‐smooth muscle actin PE‐conjugated antibody (R&D Systems # IC1420P) was added. Cells were vortexed and incubated for 30 min at room temperature in the dark. Afterwards, cells were washed two times with Flow Cytometry Permeabilization/Wash Buffer I and pellet was resuspended in 400 µL of PBS for flow cytometric analysis. Cells were analysed with BD LSR II flow cytometer (Becton Dickinson) and FlowJo (FlowJo LLC).
Flow cytometry fixation buffer
Manufactured by R&D Systems
Sourced in United States
Flow Cytometry Fixation Buffer is a laboratory reagent designed for the preparation of cells for flow cytometry analysis. It is used to fix and stabilize cell samples, preserving their morphological and antigenic properties prior to staining and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Flow cytometry permeabilization buffer, by R&D Systems (3 mentions)
Flow cytometry permeabilization wash buffer 1, by R&D Systems (3 mentions)
Flow cytometry staining buffer, by R&D Systems (2 mentions)
Purified rat igg2a κ isotype control, by BD (2 mentions)
Goat anti rat ig fitc, by BD (2 mentions)
Versene, by Thermo Fisher Scientific (2 mentions)
Anti vimentin clone rv202, by BD (2 mentions)
Anti fibronectin antibodies clone 10, by BD (2 mentions)
Pe conjugated goat anti mouse secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Facs tubes, by Avantor (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Diluent c, by Merck Group (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Goat anti mouse igg fitc, by Santa Cruz Biotechnology (1 mentions)
Mouse igg1 isotype control, by Novus Biologicals (1 mentions)
Cd44 antibody, by Novus Biologicals (1 mentions)
Lsrfortessa, by BD (1 mentions)
15 protocols using flow cytometry fixation buffer
1
Quantifying Corneal Fibroblast Activation
2
Quantitative Analysis of EV Uptake
3
HCV Core Protein Detection by Flow Cytometry
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Cells were collected by incubation with Accumax™ Cell Detachment Solution (Sigma), pelleted (300xg for 5 minutes), washed twice with Flow cytometry staining Buffer (R&D Systems) and blocked with Human Fc Blocking Solution (R&D Systems) for 15 minutes at 4°C. Cell viability was established with the LIVE/DEAD Fixable Aqua Dead cell stain kit (Life Technologies), following manufacturer instructions.
For HCV core staining, fixation was carried out by using Flow cytometry Fixation Buffer (R&D Systems) and incubating the cells for 10 minutes at room temperature. Permeabilization was performed with Flow cytometry permeabilization Buffer (R&D Systems). After an incubation of 15 minutes at room temperature, cells were stained with 1 μg (per 106 cells) of mouse anti-HCV core monoclonal antibody (Pierce, Thermo Scientific) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies) diluted 1/2000. All flow cytometry studies were performed on a FACS Calibur cytometer (BD Biosciences, San Diego, CA, USA) with data analyses conducted using FlowJo 7.6.3 software (Tree Star, Inc., Ashland, OR, USA).
For HCV core staining, fixation was carried out by using Flow cytometry Fixation Buffer (R&D Systems) and incubating the cells for 10 minutes at room temperature. Permeabilization was performed with Flow cytometry permeabilization Buffer (R&D Systems). After an incubation of 15 minutes at room temperature, cells were stained with 1 μg (per 106 cells) of mouse anti-HCV core monoclonal antibody (Pierce, Thermo Scientific) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies) diluted 1/2000. All flow cytometry studies were performed on a FACS Calibur cytometer (BD Biosciences, San Diego, CA, USA) with data analyses conducted using FlowJo 7.6.3 software (Tree Star, Inc., Ashland, OR, USA).
4
Flow Cytometric Analysis of RHAMM and CD44 in LAD2 Mast Cells
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LAD2 MCs were incubated for 1 h in normoxia (21% O2) or hypoxia (1% O2) and fixed under the same conditions for 5 min in 500 μL of flow cytometry fixation buffer (R&D Systems). After fixation, MCs were washed in FACS buffer containing 1 × PBS, 1% BSA, and 0.1% sodium azide and were subsequently resuspended in 100 μL of this buffer. Next, MCs were incubated for 30 min with primary nonspecific antibody (mouse IgG1 isotype control, Novus Biologicals) at a 1:1000 dilution, with primary antibody against the RHAMM receptor (RHAMM/CD168 antibody, Novus Biologicals) at a 1:500 dilution followed by incubation in the dark for 30 min at 4 °C with fluorescence-labeled secondary antibody (goat anti-mouse IgG-FITC, Santa Cruz Biotechnology) at a 1:400 dilution, with primary nonspecific antibody (purified rat IgG2a κ isotype control, BD Pharmingen) at a 1:500 dilution, and with primary antibody against the CD44 receptor (CD44 antibody, Novus Biologicals) at a 1:500 dilution, followed by fluorescence-labeled secondary antibody (goat anti-rat Ig-FITC, BD Pharmingen) at a 1:500 dilution. Finally, MCs were washed with FACS buffer and resuspended in 1 mL of FACS buffer. Flow cytometry analysis was performed using a BD LSR Fortessa™ (BD Biosciences).
5
Multimarker Flow Cytometry Analysis
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Cells were harvested using the non‐enzymatic dissociation buffer Versene (Life Technologies) and collected by centrifugation. Cells were exposed to an anti‐CD324 antibody (E‐cadherin, clone 180224, R & D Systems, Minneapolis, MN). For intracellular staining, cells were exposed to Flow Cytometry Fixation Buffer (R & D Systems) for 20 min at 4 °C in the dark, followed by the addition of Flow Cytometry Permeabilization buffer for 5 min at room temperature (R & D Systems). Cells were exposed to anti‐Vimentin (clone RV202) and anti‐Fibronectin antibodies (clone 10, BD Biosciences, San Jose, CA, used also for western blot) all at optimized antibody dilutions. Primary antibodies were recognized by PE‐conjugated goat anti‐mouse secondary antibodies (Life Technologies) on an LSR II Flow Cytometer (BD Biosciences).
6
Integrin Expression in Hypoxia
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Cells were fixed in normoxic (21% O2) or hypoxic (1% or 5% O2) conditions for 5 minutes in 500 μL of flow cytometry fixation buffer (R&D Systems) and washed in stain buffer containing BSA (BD Pharmingen). Cells were resuspended in 100 μL of stain buffer and incubated for 45 minutes without antibodies or with nonspecific fluorescence labeled antibody (Mouse IgG1 κ Isotype Control-Alexa Fluor 647, BD Pharmingen) at 1:20 dilution, with fluorescence labeled antibody against integrin α5 (Mouse Anti-Human CD49e-Alexa Fluor 647, BD Pharmingen) at 1:20 dilution, with primary nonspecific antibody (Purified Rat IgG2a κ Isotype Control, BD Pharmingen) at 1:25 dilution or with primary antibody against integrin β1 (Purified Rat Anti-Human CD29, BD Pharmingen) at 1:25 dilution followed by incubation for 45 minutes with fluorescence labeled secondary antibody (Goat Anti-Rat Ig-FITC, BD Pharmingen) at 1:50 dilution. Cells were washed with stain buffer and resuspended in 1 mL of stain buffer. Flow cytometry was performed using the BD LSRFortessaTM (BD Biosciences).
7
Flow Cytometry Sample Preparation
8
Cell Viability and Phenotypic Analysis
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Cell pellets were resuspended in 100 μl of 1% fetal bovine serum (FBS; VWR) and incubated with either trypan blue (Invitrogen) or LIVE/DEAD Fixable Yellow stain (Invitrogen) for 5 min at room temperature and 30 min at 37°C, respectively. Fixable Yellow samples were subsequently fixed using Flow Cytometry Fixation Buffer (R&D Systems), centrifuged, and resuspended in 1% FBS in PBS. trypan blue cell viability was measured using Countess II Automated Cell Counter (Thermo Fisher Scientific). For flow cytometry, samples were fixed in fixation buffer for 30 min at 4°C, washed with permeabilization buffer (R&D Systems), centrifuged, and incubated with fluorescent antibodies (table S2) in permeabilization buffer at 4°C for 1 hour. Samples were then washed with 10% FBS in PBS, centrifuged, and resuspended in 10% FBS in PBS. Appropriate negative internal controls were used. Data acquisition was done with a NovoCyte 3000 (ACEA Biosciences) and data analysis with NovoExpress.
9
Cell Labeling and Flow Cytometry Protocol
10
MuV Infection Inhibition by CD437
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IFNAR1-KO A549/hSLAM cell lines were seeded in 6 well plate 1 × 106 cells per well and infected with MuV at a MOI of 2. After 18 h of cultivation with different concentrations of CD437 (mock, 1, 3, and 10 μM), the cells were harvested using 0.25% trypsin (Nacalai Tesque Co.). The cells were fixed and re-suspended using Flow Cytometry Fixation Buffer and Flow Cytometry Staining Buffer (R and D systems, MN, United States), and GFP expression was measured using BD FACSLyric (Becton, Dickinson and Company, NJ, United States).
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