Rat GEC monolayers incubated with Kgp or TCLK-treated Kgp were gently dispersed and resuspended at a final concentration of 3×106 cells/ml. After washing with PBS, cells were fixed with OptilyseC (Becton Dickinson, Franklin Lakes, NJ) containing 0.2% Triton X-100 (Sigma-Aldrich). Next, cells were washed with 0.2% Triton X-100 in PBS (washing buffer) and incubated with anti-cytokeratin-6 antibody (20 μg/ml) or with the same concentration of non-immune serum for 1 h in washing buffer, followed by incubation with FITC-conjugated secondary antibody (ICN Pharmaceuticals, Aurora, OH) for 30 min. Fluorescence was analyzed with a FAC-Scan analyzer (Beckman Coulter, Fullerton, CA).
Optilysec
Manufactured by BD
Sourced in United States
The OptilyseC is a lab equipment product designed for cell lysis. It facilitates the breakdown of cell membranes to release cellular contents for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using optilysec
1
Immunofluorescence Analysis of Rat GEC Cells
2
RAGE Expression Analysis in OLC-1 Cells
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OLC-1 monolayers were gently dispersed and resuspended at a final concentration of 3 × 106 cells/mL and FACS was performed as described previously [38 ] with slight modifications. After washing with PBS, cells were fixed with OptilyseC (Becton Dickinson, Franklin Lakes, NJ, USA). Next, cells were washed with PBS and incubated with the RAGE Ab or isotype-matched control (2 μg/mL), at 4°C for 1 h, followed by the fluorescein isothiocyanate (FITC-) conjugated secondary Ab (ICN Pharmaceuticals, Aurora, OH, USA) for 30 min in the dark. Fluorescence was analyzed with a FACScan analyzer (Beckman Coulter, Fullerton, CA, USA).
3
Flow Cytometry Enumeration of CD4+ Cells
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The FACSLyric was used as a flow cytometer for counting CD4 in people living without HIV and in people living with HIV. A tube containing a mixture of 200 µL of peripheral blood, 5 µL of BB515-CD45 (BD Biosciences, Franklin Lakes, NJ, USA), and 5 µL of PE-CD4 (BD Biosciences, NJ, USA) was incubated at room temperature in the dark for 15 min. After staining the cells, the mixture was added to 1 mL of Lysing solution OptiLyse C (BD Biosciences, NJ, USA) and then incubated for 10 min. The cells were then centrifuged at 2000× g rpm for 5 min after adding 5 mL of PBS (Phosphate-buffered saline, pH 7.4). The supernatant was removed and the cells resuspended in 1 mL PBS. The resuspended sample (1 mL) was then loaded into the flow cytometer and the CD4/CD45 ratio calculated using flow cytometry. The CD4 gating strategy was performed with reference to a previous study [22 (link)].
4
Isolation of PBMCs from Whole Blood
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The blood samples were taken from healthy adult volunteers. One milliliter of whole blood was incubated with 5 mL of the hemolytic agent (Beckman Coulter, Optilyse C) for 10 min and additional 15 min after adding 10 mL of CellWash (BD Biosciences). Then, the samples were centrifuged at 430 g for 5 min. After the removal of the supernatants, the pellets were suspended with 5 mL of CellWash and used as PBMCs. PBMCs and whole blood cells were diluted with phosphate buffer saline to adjust a cell count of 107 cells/mL and subjected to the SRS imaging flow cytometer within 5 h after the sample preparation. This procedure was performed at room temperature (18–25 °C). This study was approved by the Institutional Ethics Committee of Faculty of Medicine, the University of Tokyo (No. 11049-5) and conducted according to the Declaration of Helsinki. All volunteers provided written informed consent.
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