Cells were blocked in PBS plus 2% FCS supplemented with 20% human serum, then labelled with antibodies (eBiosciences, Hatfield UK), for CD4+ T cells (PE-CD14 or PE-CD4), for B cells (FITC-CD3 or FITC CD19) and for monocytes (FITC-CD3 or FITC-CD14). Flow cytometry was performed using a Guava 8HT (Millipore, UK) running Guavasoft 2.5.
Guava 8ht
Manufactured by Merck Group
Sourced in United Kingdom, United States
The Guava 8HT is a compact and user-friendly flow cytometer designed for high-throughput cell analysis. It features an 8-color detection system and is capable of analyzing up to 96 samples in a single run. The Guava 8HT provides reliable and efficient data acquisition for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Gauvasoft software, by Merck Group (4 mentions)
Cd4 pe, by Thermo Fisher Scientific (1 mentions)
Cd14 pe, by Thermo Fisher Scientific (1 mentions)
Isotype control abs, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Cd16 fitc, by BD (1 mentions)
Cd14 percp, by BD (1 mentions)
Cd80 allophycocyanin apc, by BD (1 mentions)
Phycoerythrin conjugated antibodies, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Ic fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Kaluza, by Beckman Coulter (1 mentions)
Cd4 pe, by BioLegend (1 mentions)
Cd25 apc cy7, by BioLegend (1 mentions)
Anti mouse antibodies, by BioLegend (1 mentions)
Il17a apc, by BioLegend (1 mentions)
Il 6 fitc, by BioLegend (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Daf fm, by Thermo Fisher Scientific (1 mentions)
Infinite m200 plate reader, by Tecan (1 mentions)
15 protocols using guava 8ht
1
Immunophenotyping of Blood Cell Subsets
2
PD-L1 Binding Assay with Atezolizumab
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Example 6
Flow cytometry was performed to access binding of atezolizumab and atezolizumab bound Abraxane to the ligand, PD-L1. The PD-L1 positive melanoma cell line, C8161 was used for this experiment. AA130 was made as described above and an aliquot of the nanoparticles was spun at 6000 rpm for 10 minutes to remove any unbound atezolizumab. C8161 cells were stained with FITC labeled isotype control and anti-human PD-L1 as negative and positive controls, respectively. The C8161 cells were incubated for 30 minutes with ABX and atezolizumab alone and the AA130 nanoparticle. After the incubation the cells were labeled with FITC labeled anti-human PD-L1 for 30 minutes and washed with FACS buffer (1×PBS+0.5% BSA and 0.05% Na azide). After washing, the cells were analyzed by flow cytometer on the Guava 8HT and data analysis performed with Gauvasoft software (Millipore).
C8161 cells were pre-treated with isotype control antibody (
3
Analyzing PD-L1 Binding of Atezolizumab
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Example 3
Flow cytometry was performed to access binding of atezolizumab and atezolizumab bound Abraxane to the ligand, PD-L1. The PD-L1 positive melanoma cell line, C8161 was used for this experiment. AA130 was made as described above and an aliquot of the nanoparticles was spun at 6000 rpm for 10 minutes to remove any unbound atezolizumab. C8161 cells were stained with FITC labeled isotype control and anti-human PD-L1 as negative and positive controls, respectively. The C8161 cells were incubated for 30 minutes with ABX and atezolizumab alone and the AA130 nanoparticle. After the incubation the cells were labeled with FITC labeled anti-human PD-L1 for 30 minutes and washed with FACS buffer (1×PBS+0.5% BSA and 0.05% Na azide). After washing, the cells were analyzed by flow cytometer on the Guava 8HT and data analysis performed with Gauvasoft software (Millipore).
C8161 cells were pre-treated with isotype control antibody (
4
Multiparametric Flow Cytometry Analysis
5
Bone Marrow and Blood Analysis
6
Dose-dependent Cytotoxicity of ABX and AA130 in C8161 Melanoma Cells
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Example 4
C8161 melanoma cells were exposed to ABX and AA130 at paclitaxel concentrations from 0 to 200 μg/mL overnight to determine cell toxicity. The cells were also incubated with EdU, a thymidine analog. The next day the cells were harvested, fixed with 2% paraformaldehyde and permeabolized with 1% saponin. After permeabolization the cells were incubated for 30 minutes with a FITC labeled anti-EdU antibody to determine the percentage of cells proliferating. After washing, the cells were analyzed by flow cytometer on the Guava 8HT and data analysis performed with Gauvasoft software (Millipore). The proliferation index was calculated by normalization to an untreated positive control.
7
Multicolor Flow Cytometry for Cell Characterization
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The fluorochrome-labeled monoclonal antibodies used in this study included: antibodies against CD14-percp (BD), CD16-FITC (BD Pharmingen), IL-6-phycoerytherin (PE, BD Pharmingen), CD80-allophycocyanin (APC, BD Pharmingen), CD86-PE, and isotype control antibodes (BD Pharmingen). Cells were identified by their forward and side scatter characteristics and were analyzed by flow cytometry on a Guava 8HT flow cytometer (Millipore, Billerica, MA).
8
Flow Cytometry Analysis of Adipose-Derived Stem Cells
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The stromal vascular fraction samples were analyzed for the presence of adipose-derived mesenchymal stem cells using flow cytometry (Guava 8HT; Millipore, Billerica, Mass.). To obtain enough cells for flow cytometry, cultured stromal vascular fractions were used at passage 0 after cells reached 75 percent confluence. Single-cell suspensions from passage 0 cells were stained with mouse anti-human CD14–fluorescein isothiocyanate (1 μg/ml), CD19–fluorescein isothiocyanate (1 μg/ml), CD90-fluorescein isothiocyanate (1 μg/ml), CD105–fluorescein isothiocyanate (1 μg/ml), CD73-phycoerythrin (1 μg/ml), CD45-phycoerythrin (1 μg/ml), CD34-phycoerythrin (1 μg/ml), and CD13-phycoerythrin (1 μg/ml) using standard protocols.45 (link) Mouse fluorescein isothiocyanate– or phycoerythrin-conjugated mouse immunoglobulin G1 (1 μg/ml) was used as an isotype control. Fluorescein isothiocyanate–conjugated antibodies were purchased from Serotec (Raleigh, N.C.) and phycoerythrin-conjugated antibodies were purchased from BD Biosciences (San Jose, Calif.).
9
Multiparametric Flow Cytometry of PBMCs
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Freshly thawed or CMV-stimulated PBMC were washed in staining medium and incubated for 30 min at 4°C with surface markers. For intracellular Granzyme B, TGFβ, IL-35 and IL-10 staining, PBMC were then permeabilized and fixed (Cytofix/Cytoperm; BD Biosciences) for 20 min at 4°C, washed and incubated with the appropriate mAbs. For intranuclear FoxP3, cells were permeabilized and fixed with IC Fixation and permeabilization buffer (eBiosciences) for 1 hour at 4°C, washed and incubated with the mAb. At the completion of the staining procedure, cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences) in PBS and data were acquired with Guava 8HT (Millipore) or Gallios (Beckman Coulter). Results were analyzed with FlowJo (Treestar) or Kaluza (Beckman Coulter).
10
Dose-Dependent Toxicity of ABX and AA130 on C8161 Melanoma Cells
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Example 7
C8161 melanoma cells were exposed to ABX and AA130 at paclitaxel concentrations from 0 to 200 μg/mL overnight to determine cell toxicity. The cells were also incubated with EdU, a thymidine analog. The next day the cells were harvested, fixed with 2% paraformaldehyde and permeabolized with 1% saponin. After permeabolization the cells were incubated for 30 minutes with a FITC labeled anti-EdU antibody to determine the percentage of cells proliferating. After washing, the cells were analyzed by flow cytometer on the Guava 8HT and data analysis performed with Gauvasoft software (Millipore). The proliferation index was calculated by normalization to an untreated positive control.
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