Depending on the experiments, cell populations were labeled with combinations of the following antibodies: Live/Dead stain (APC-CY7; Invitrogen), CD4 (clone SK3; BD), CD8 (clone RPA-T8; BD), p24 (clone KC57-RD1; Coulter Clone), MIP-1β (clone 24006; R&D Systems), and CD107a (clone H4A3; BD). The intracellular staining assay was performed as described previously (25 (link)), with some modifications. Briefly, CD107a was added to the culture with CD8+ T cells and incubated for an additional 3 h in the presence of brefeldin A (10 μg/ml) and GolgiStop. Cells were then stained first with Live/Dead stain and for surface markers of CD4 and CD8 and fixed with 1% formaldehyde overnight. The following day, the cells were permeabilized with buffer containing saponin and stained for p24 and/or MIP-1β. All samples were fixed for 2 h with 1% formaldehyde before being acquired on an LSRII flow cytometer. Data were analyzed with FlowJoV (Tree Star Inc.).
Flowjov
Manufactured by Tree Star
Sourced in United States
FlowJoV is a powerful data analysis software for flow cytometry. It provides advanced tools for visualization, compensation, gating, and statistical analysis of flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 clone rpa t8, by BD (1 mentions)
Cd4 clone sk3, by BD (1 mentions)
Cd107a clone h4a3, by BD (1 mentions)
Diva software, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Alexa 647, by BD (1 mentions)
Apc cy7, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Apc cy7, by BD (1 mentions)
4 protocols using flowjov
1
Multicolor Flow Cytometry Protocol
2
Cell Cycle Analysis by Flow Cytometry
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Cells were cultivated for six days with or without 1 µM/5 µM Genz as described above. Cells were washed with PBS, trypsinized and transferred to 15 mL conical tubes. The tubes were centrifuged at 1500 rpm and supernatant decanted. The residual amount of liquid was carefully removed with a pipette. The cells were suspended in 250 µL PBS by gently pipetting up and down for approximately five times. Ice cold methanol (700 µL, precooled at −20 °C) was added dropwise while slowly vortexing the tubes. The cells were incubated at 4 °C for at least one hour. Approximately 106 cells were added to 4 mL precooled PBS with 1% FCS into FACS tubes and centrifuged at 1500 rpm for 5 min. Cells were stained with 400 µL PI-solution + RNAse A (0.01 mg/mL PI, 0.25 mg RNAse/mL in PBS/1% FCS). Cells were incubated at RT in the dark for 30 min and immediately measured by FACS in linear mode on a FACS Canto with Diva software (BD, East Rutherford, NJ, USA). Analysis of the data was performed using FlowJo V (Tree Star, Flow Cytometry Analysis Software, Ashland, OR, USA).
3
Single-cell oral mucosal tissue analysis
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Biopsy preparation into a single cell suspension of oral mucosal tissues was performed as previously described [58 (link)]. Data were analysed with FlowJo software (FlowJo v10.8; TreeStar). Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS, version 25.0). The Kolmogorov–Smirnov test and normal Q–Q plots were used for tests of normality. The Levene test was used to determine the homogeneity of variance. One-way ANOVA and Student’s t-test for parametric statistical tests, and the Kruskal–Wallis rank sum test and Wilcox rank sum test for non-parametric statistical tests were selected based on the data characteristics of normality and homogeneity of variance.
4
Characterization of CTL Dysfunction Markers
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To characterize potential markers associated with CTL dysfunction in response to LRA-reactivated cells, we exposed CTL1 and CTL2 to cognate HIV-1 antigen over nine weeks. To do so, we stimulated CTL with a 1:1 mixture of irradiated autologous B-cell lines (BCLs) pulsed with cognate HIV peptide (10 μg/ml) and irradiated PBMCs from three healthy HIV-seronegative donors weekly. We used flow cytometry to evaluate variations in the expression of the inhibitory receptors PD-1, TIM-3, LAG-3, and the immune-metabolic marker CD39. Briefly, cells were taken from the culture at weeks 0, 5, 7, and 9 and stained with a Live/Dead probe (APC-Cy7, Invitrogen) and surface markers CD3 (Alexa700, BD Biosciences), CD4 (APC-Cy7, BD Biosciences), CD8 (V500, BD Biosciences), PD-1 (BV421, BD Biosciences), TIM-3 (Alexa 647, BD Biosciences), LAG-3 (PE, BD Biosciences), and CD39 (FITC, BD Biosciences) and incubated at room temperature for 25 min. Samples were washed twice with 1X PBS, fixed in 1% formaldehyde, and acquired on an LSR Fortessa. Data were analyzed with FlowJoV (Tree Star Inc.). Patterns of co-expression of PD-1, LAG-3, TIM-3, and CD39 were analyzed using Pestle and SPICE v5 software (55 (link)).
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