The analyses of the second cohort (Table 2 ) included evaluation of monocyte and granulocyte subpopulations. Immune cell subpopulations were analyzed by flow cytometry after FACS staining which was performed within 1 h of blood withdrawal on day of admission and day 1, 3 and 5 post stroke (LSRII, BD Bioscience) [anti-HLA-DR Alexa Flour 488; clone: L243; anti-CD11b Brilliant Violet 421, clone: LM2; anti-CD14 PerCP/Cy5.5, clone: 63D3; anti-CD16 Brilliant Violet 650, clone: 3G8; anti-CD62Ligand PE-Cy7, clone: DREG-56; and anti-CD32-PE, clone: FUN-2 (Biolegend)]. Dead cells were excluded from analysis by the Zombie NIR TM Fixable Viability Kit (Biolegend). The results were evaluated using FlowJo Software 7.6.5 (Tree Star Inc.). The percentage of cells expressing a specific activation marker was determined as well as the amount of the specific marker per cell as defined by the mean fluorescence intensity (MFI). Fluorescence minus one controls (FMO) were used to distinguish different monocyte and granulocyte populations.
Anti cd14 percp cy5
Manufactured by BioLegend
Sourced in United States, United Kingdom
Anti-CD14 PerCP/Cy5.5 is a fluorochrome-conjugated antibody that binds to the CD14 cell surface receptor. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed predominantly on monocytes and macrophages. The PerCP/Cy5.5 fluorochrome allows for multicolor flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti hla dr alexa flour 488, by BD (2 mentions)
Anti cd62ligand pe cy7, by BioLegend (2 mentions)
Anti cd32 pe, by BioLegend (2 mentions)
Zombie nirtm fixable viability kit, by BioLegend (2 mentions)
Anti cd11b brilliant violet 421, by BioLegend (1 mentions)
Facsdiva v6, by BD (1 mentions)
Anti cd19 fitc, by BD (1 mentions)
Anti cd56 fitc, by BD (1 mentions)
Anti cd3 fitc, by BD (1 mentions)
Anti hla dr pe, by BD (1 mentions)
Anti cd11b apc, by BioLegend (1 mentions)
Anti cd20 fitc, by BD (1 mentions)
Anti cd57 fitc, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Anti cd11c apc, by BioLegend (1 mentions)
Recombinant human il 4, by R&D Systems (1 mentions)
Anti cd83 pe, by BioLegend (1 mentions)
Anti cd1a fitc, by BioLegend (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti cd14 percp cy5
1
Flow Cytometry Analysis of Immune Cells
2
Phenotypic Analysis of Frozen PBMCs
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Frozen PBMCs from HLA-A2 patients (at least 30 × 106 cells at day 0 and 6 days after vaccination) were thawed as previously described.50 Cells were washed twice with ice-cold PBS and were incubated with Human TrueStainFcXTM Fc Receptor Blocking Solution (BioLegend cat. no. 422301) at room temp. for 5 min to block non-specific binding of antibodies. Next, cells were labeled with recommended amounts of anti-CD3-FITC (BD Biosciences cat no. 555916), anti-CD19-FITC (BD Bioscience cat no. 555412), anti-CD20-FITC (BD Bioscience cat no. 555622), anti-CD57-FITC (BD Bioscience cat no. 555619), anti-CD56-FITC (BD Bioscience cat no. 562794), anti-HLA-DR-PE (BD Bioscience cat no. 555812), anti-CD14-PerCP-Cy5.5 (BioLegend cat no. 301823) and anti-CD11b-APC (BioLegend cat no. 301310) at 4°C in the dark for 30 min. Labeled cells were washed twice with PBS, resuspended in 0.4 ml PBS, and immediately analyzed on a FACSCanto flow cytometer. Data acquisition was performed using BD FACSDiva v.6.1.2.
3
Monocyte and Granulocyte Immunophenotyping
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EDTA blood was sampled and processed within 2 h to examine monocytes and granulocytes. After a red blood cell lysis using ACK lysing buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), cells were stained by Zombie NIRTM Fixable Viability Kit (BioLegendTM) on ice for 15 min to distinguish dead and alive cells, followed by a second staining with the different cell surface antibodies for 10 min on ice. Subpopulations were analyzed by flow cytometry (LSRII, BD Bioscience) [anti-HLA-DR Alexa Flour 488, anti-CD11b Brilliant Violett 421, anti-CD14 PerCP/Cy5.5, anti-CD16 Brilliant Violett 650, anti-CD62Ligand PE-Cy7, anti-CD32-PE (Biolegend)]. Death cells were distinguished by Zombie NIR TM Fixable Viability Kit (Biolegend). 200.000 events were gathered per single cell gate.
The results were evaluated using FlowJo Software 7.6.5 (Tree Star Inc.). The percentage of cells expressing a specific activation marker was determined as well as the amount of the specific marker on cell surface as defined by the mean fluorescence intensity (MFI). For the differentiation of monocytes and granulocyte subpopulation as well as activation marker fluorescence minus one controls (FMO) were used. CD14dim monocytes and CD16dim neutrophil population was distinguished by gating the 25th percentile of main monocyte and neutrophil population, respectively (seeSupplementary Figures 1 , 2 for gating strategy).
The results were evaluated using FlowJo Software 7.6.5 (Tree Star Inc.). The percentage of cells expressing a specific activation marker was determined as well as the amount of the specific marker on cell surface as defined by the mean fluorescence intensity (MFI). For the differentiation of monocytes and granulocyte subpopulation as well as activation marker fluorescence minus one controls (FMO) were used. CD14dim monocytes and CD16dim neutrophil population was distinguished by gating the 25th percentile of main monocyte and neutrophil population, respectively (see
4
Generation of Dendritic Cells from Monocytes
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DCs were generated from monocytes isolated from PBMC. Purified monocytes (1 ×
106 cells/mL) were resuspended in RPMI 1640 containing human
recombinant IL-4 (80 ng/mL) and GM-CSF (80 ng/mL) (R&D Systems, USA) for 7
days at 37°C and 5% CO2 [29 (link),
30 (link)]. Then, cells were incubated with
mAbs (Biolegend, USA) anti-CD14-PerCP-Cy 5.5 (0.3 μL), anti-CD1a-FITC (1 μL),
anti-CD83-PE (1 μL) and anti-CD11c-APC (1 μL) for 30 min. A Fluorescence Minus
One (FMO) control was included.
This phenotyping protocol was performed to assure the cell differentiation and
analyzed in a flow cytometer model FACS CaliburTM (BD Becton Dickinson and
Company, USA). A total of 50.000 events were acquired and the expression of
following cell surface markers was analyzed:
CD14low/CD1ahigh/CD11chigh/CD83low[31 (link)].
DCs were incubated with propolis alone or in combination with MAGE-1 and RA for
48 h.
106 cells/mL) were resuspended in RPMI 1640 containing human
recombinant IL-4 (80 ng/mL) and GM-CSF (80 ng/mL) (R&D Systems, USA) for 7
days at 37°C and 5% CO2 [29 (link),
30 (link)]. Then, cells were incubated with
mAbs (Biolegend, USA) anti-CD14-PerCP-Cy 5.5 (0.3 μL), anti-CD1a-FITC (1 μL),
anti-CD83-PE (1 μL) and anti-CD11c-APC (1 μL) for 30 min. A Fluorescence Minus
One (FMO) control was included.
This phenotyping protocol was performed to assure the cell differentiation and
analyzed in a flow cytometer model FACS CaliburTM (BD Becton Dickinson and
Company, USA). A total of 50.000 events were acquired and the expression of
following cell surface markers was analyzed:
CD14low/CD1ahigh/CD11chigh/CD83low[31 (link)].
DCs were incubated with propolis alone or in combination with MAGE-1 and RA for
48 h.
5
Multiparametric Flow Cytometry of Immune Cells
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Zombie UV™ Fixable Viability Kit, anti-CD45 BV570, anti-CD3 BV605, anti-CD4 BV421, anti-CD8 BV650, anti-CD4 APC-Fire750, anti-CD73 PE/Dazzle 594, anti-CD56 PE, anti-CD14 PerCP/Cy5.5, anti-CD16 Alex Fluor 700, anti-CD19 APC-Fire750, anti-HLA-DR PE-Cy5, anti-PD-1 BV421, anti-CD16 PE, anti-IFNγ A700, anti-IL-13 APC, anti-IL-5 APC, anti-TNFα Alexa Fluor 488, and anti-IL-21 PE were purchased from BioLegend (London, UK). Anti-CD25 BB515, anti-CD4 BUV496, anti-CD39 BV711, anti-Perforin Alexa Fluor 647, anti-Granzyme B Alexa Fluor 700, anti-Ki-67 Alexa Fluor 700, anti-CD45RA PE, anti-IL-2 PerCP/Cy5.5, and Fixation/Permeabilization Solution Kit were purchased from Becton Dickinson. Anti-FOXP3 APC, anti-CD56 APC-eFluor780, and eBioscience™ (San Diego, CA, USA) Foxp3/Transcription Factor Staining Buffer Set were purchased from eBioscience TermoFisher scientific. Anti-TIM-3 APC was purchased from R&D systems. Anti-CD137 PE and FcR blocking reagent were purchased from Miltenyi (Bergisch Gladbach, Germany).
Peptides for the PSA, PSMA, PSCA, and PAP pools (Supplementary Table S1 ) were synthetized and lyophilized by the Peptide and Tetramer Core Facility of the Department of Oncology at UNIL-CHUV (Lausanne, Switzerland), and 5T4 pools (Supplementary Tables S2 and S3 ) were synthetized and lyophilized by JPT Peptide Technologies.
Peptides for the PSA, PSMA, PSCA, and PAP pools (
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