Vero and SK-N-SH cells were cultured in 24-well plates (1 × 105 cells/well) overnight and infected with HSV-1 GFP (MOI = 2) at 37 °C for 2 h. The cells were washed three times with PBS, and the medium was replaced with complete DMEM. QA was added at 50 and 100 µg/mL to each well, and the cells were incubated at 37 °C for 48 h. As a positive control, acyclovir (ACV) was used. HSV-GFP expression was measured under a fluorescence microscope (Nikon ECLIPSE Ti-U, Nikon Co., Japan). Vero and SK-N-SH cells were harvested and resuspended in 0.5 mL PBS containing 2% FBS and fixed in suspension with 4% paraformaldehyde (PFA). The cells were washed three times with PBS and stored at 4 °C until analysis using a CytoFLEX flow cell counter (Beckman Coulter Inc., Pasadena, CA, USA) and FlowJo software.
Cytoflex flow cell counter
Manufactured by Beckman Coulter
Sourced in United States
The CytoFLEX flow cell counter is a laboratory instrument designed to analyze and quantify cells, particles, and other biological entities. It uses flow cytometry technology to detect and measure various properties of individual cells or particles as they pass through a laser beam within the instrument. The CytoFLEX is capable of capturing and analyzing multiple parameters, including size, granularity, and fluorescence, providing researchers with detailed information about the sample under examination.
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Lab products found in correlation
Collagenase 4, by Merck Group (2 mentions)
Eclipse ti u, by Nikon (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
Ammonium chloride potassium lysing buffer, by Lonza (1 mentions)
Apc anti mouse cd8a, by BioLegend (1 mentions)
Red blood cell lysis buffer, by Lonza (1 mentions)
Percp cy5.5 rat igg2b, by BioLegend (1 mentions)
Percp cyanine5.5 anti mouse cd45, by BioLegend (1 mentions)
Apc rat igg2a, by BioLegend (1 mentions)
5 protocols using cytoflex flow cell counter
1
Evaluating QA Inhibition of HSV-1 Infection
2
Tumor Infiltrating Lymphocytes Isolation
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Tumors from control, Abseff and Absineff group were selected for tumor extraction. The single-cell suspension of mouse splenocytes was prepared by filtering through a 100-mm and 40-mm cell strainer (SPL Life Sciences, Pocheon , Korea) while red blood cells (RBCs) were removed with ammonium-chloride-potassium lysing buffer (Lonza, Basel, Switzerland). Further, tumors were digested with collagenase IV (0.2 mg/mL, Sigma-Aldrich) and DNase I (0.02 mg/mL, Sigma-Aldrich) for 30 min. Tumor tissues were then squashed through 70 m cell strainers with a syringe. TILs were extracted from the central layer of a 40–80% Percoll (Sigma-Aldrich) gradient centrifugation (run at 800 g for 20 min at room temperature without deceleration). CytoFLEX flow cell counter (Beckman) was used to examine TILs such as CD4+/CD5+, CD3+, CD8+/CD3+, CD25+/CD4+, CD45+, CD103+/CD8+, CD39+/CD8+, CD11c+, F4/80+, NK1.1+, CD86+, CD68+, and CD206+.
3
Antiviral Efficacy of RVS and RVSE
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MDCK or A549 cells were cultured in 24-well plates (1 × 105 cells/well) for 18 h. A/PR/8/34 (MOI = 1) was mixed with different concentrations of RVS and RVSE (0, 12.5, 25, 50, and 200 μg/mL), and the mixtures were incubated at 37°C for 1 h. MDCK and A549 cells were infected with these mixtures at 37°C for 2 h. Subsequently, the virus was removed, and the cells were washed three times with PBS, and the medium was replaced by complete DMEM. Cells were incubated for 24 h at 37°C with 5% CO2. Reduction of viral infection was determined by measuring GFP expression using flow cytometry. MDCK or A549 cells were harvested and resuspended in 1 mL of PBS containing 2% FBS and fixed in suspension with 4% paraformaldehyde. The cells were washed three times with PBS and stored at 4°C until analysis with a CytoFLEX flow cell counter (Beckman). We analyzed data using FlowJo software.
4
MDCK Cell Antiviral Assay with GHE and GN
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MDCK cells were cultured in 12-well plates (5 × 105 cells/well) for 18 h. Next, A/PR/8/34 (MOI = 1) was mixed with different concentrations of GHE (100 and 200 μg/mL) and GN (10 and 100 μM), and the mixtures were incubated at 37 °C for 1 h. Then, MDCK cells were infected with these mixtures at 37 °C for 2 h. Subsequently, the virus was removed, and the cells were washed three times with PBS and the medium was replaced by complete DMEM. The cells were incubated for 24 h at 37 °C with 5% CO2. The reduction of viral infection effect was determined by measuring GFP expression using flow cytometry. MDCK cells were harvested and resuspended in 1 mL of PBS containing 2% FBS and fixed in suspension with 4% paraformaldehyde. The cells were washed three times with PBS and stored at 4 °C until analysis with a CytoFLEX flow cell counter (Beckman). We analyzed the data using FlowJo software.
5
Tumor Dissociation and Flow Cytometry
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Tumor tissues were harvested in RPMI Medium 1640 with 10% heat-inactivated FBS, 1% penicillin and streptomycin (all from HyClone™). Tumors were minced and incubated for 30 min in DPBS (Well-gene) with 0.5 mg/ml Collagenase IV (Sigma) at 37 °C. Tissues were squeezed through a 100 μm cell strainer (# 93100, SPL), re-filtered through a 70 μm cell strainer (# 93070, SPL) subjected for 5 min to Red Blood Cell Lysis Buffer (#10-548E, Lonza). Flow cytometry was performed to determine cell surface antigen expression by 30-min incubation on ice with pertinent antibodies. The following monoclonal antibodies were used: mouse-specific monoclonal antibodies used were PerCP/Cyanine5.5 anti-mouse CD45 (#103131, BioLegend), APC-anti-mouse CD8a (#100712, BioLegend) and corresponding isotype control mAbs PerCP/Cy5.5 Rat IgG2b (#400632, BioLegend) and APC Rat IgG2a (#400512, BioLegend). The cells were washed thrice with PBS containing 2% FBS and fixed in suspension with 4% paraformaldehyde and stored at 4 °C until analysis with a CytoFLEX flow cell counter (Beckman). We analyzed the data using FlowJo software.
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