Single-cell suspensions from rectal pinches were prepared as previously described30 (link). Rectal intraepithelial lymphocytes (IEL) and lamina propria(LP) were collected and subjected to flow cytometry analysis. The single-cell suspensions were first incubated with Fc Receptor blocking reagent (Miltenyi Biotec), followed by staining with viability dye (Invitrogen). The antibody mixtures were then incubated as previously described27 (link). For immune activation, the following antibodies were used: CD45-PerCP, CD3-PE-Cy7, CD4-BV605, CD8-APC-Cy7, CD14-V450, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (BD Pharmingen); CD69-Alexa Fluor 700 (Biolegend); and CD38-FITC (STEMCELL Technologies). For detection of Treg and MDSCs, the following antibody mixture were used: CD45-PerCP/Cy5.5, CD3-PE-Cy7, CD4-BV605, CD8-BV785, lin 1-FITC (BD Pharmingen), FOXP3-APC (eBioscience), HLA-DR-APC-Cy7, CD11b-PE-Cy5, CD14-BV711, CD8-BV785, CD25-BV421, CD15-Alexa700 (Biolegend), CD33-PE (Milteny). An LSRII flow cytometer was used for data acquisition. FlowJo software (Tree Star Inc.) was used for data analyses.
Cd4 bv605
Manufactured by BD
Sourced in United States
The CD4-BV605 is a fluorochrome-conjugated antibody used for the detection and enumeration of CD4-positive T cells in flow cytometry analysis. It provides a specific and reliable measurement of CD4+ T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 apc cy7, by BD (5 mentions)
Cd3 pe cy7, by BD (4 mentions)
Live dead aqua, by Thermo Fisher Scientific (4 mentions)
Viability dye, by Thermo Fisher Scientific (3 mentions)
Hla dr apc cy7, by BioLegend (3 mentions)
Cd11b pe cy5, by BioLegend (3 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (3 mentions)
Cd3 alexa fluor 700, by BD (3 mentions)
Cxcr5 af488, by BD (3 mentions)
Cd8 apc h7, by BD (3 mentions)
Phytohemagglutinin (pha), by Roche (3 mentions)
Ze5 cell analyzer, by Bio-Rad (3 mentions)
Cd137 apc, by BioLegend (3 mentions)
Cd3 af700, by Thermo Fisher Scientific (3 mentions)
Ox40 pe cy7, by BioLegend (3 mentions)
Human ab serum, by Gemini Bio (3 mentions)
Cd14 v500, by BD (3 mentions)
Cd3 percp cy5, by BD (3 mentions)
Cd8 bv711, by BD (3 mentions)
Cd38 pe, by BD (2 mentions)
28 protocols using cd4 bv605
1
Rectal Immune Cell Characterization
2
Rectal Immune Cell Characterization
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Single-cell suspensions from rectal pinches were prepared as previously described30 (link). Rectal intraepithelial lymphocytes (IEL) and lamina propria(LP) were collected and subjected to flow cytometry analysis. The single-cell suspensions were first incubated with Fc Receptor blocking reagent (Miltenyi Biotec), followed by staining with viability dye (Invitrogen). The antibody mixtures were then incubated as previously described27 (link). For immune activation, the following antibodies were used: CD45-PerCP, CD3-PE-Cy7, CD4-BV605, CD8-APC-Cy7, CD14-V450, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (BD Pharmingen); CD69-Alexa Fluor 700 (Biolegend); and CD38-FITC (STEMCELL Technologies). For detection of Treg and MDSCs, the following antibody mixture were used: CD45-PerCP/Cy5.5, CD3-PE-Cy7, CD4-BV605, CD8-BV785, lin 1-FITC (BD Pharmingen), FOXP3-APC (eBioscience), HLA-DR-APC-Cy7, CD11b-PE-Cy5, CD14-BV711, CD8-BV785, CD25-BV421, CD15-Alexa700 (Biolegend), CD33-PE (Milteny). An LSRII flow cytometer was used for data acquisition. FlowJo software (Tree Star Inc.) was used for data analyses.
3
Analyzing DENV-specific T Cell Responses
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Cryopreserved PBMCs were thawed and plated at ~1 × 106 cells per well in R10 medium with 0.5 μg/mL/peptide of the respective DENV-1–4 CME peptide pool. R10 containing 0.5% DMSO was used as a negative control. Cells were cultured at 37 °C for 6–7 days, and the supernatants were stored at –80 °C for the cytokine analysis. The cells were washed and stained with LIVE/DEAD Aqua as well as BV785-CD3, BV605-CD4, Alexa700-CD8, BV510-CD14, BV510-CD19, APC-Cy7-CD16, PE-Cy7-CD56, PE-CD38, and PE-Dazzle594-HLA-DR (BD Biosciences). After fixation in 4% formaldehyde, cells were permeabilized and stained with Alexa488-Ki67 (BD Biosciences). Data were collected using a BD LSRFortessa flow cytometer (BD Biosciences).
4
Multiparameter Flow Cytometry Profiling
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PBMCs were stained with different antibodies according to the manufacturer's instruction, in which antibodies used for the experiments included APC/ CY7-CD8, BV605-CD4, PE/CY7-CD25, FITC-PD-1, PE-Tim-3 or isotype-matched control IgG. All the antibodies and isotype controls were purchased from BD PharMingen San Diego, CA, USA. After staining, cells were washed twice with PBS and were subjected to flow cytometry analysis using a FACS Foterassa (BD, San Diego, CA, USA). Analyses of flow cytometry were performed by FlowJo software.
5
Multiparametric Imaging Flow Cytometry of Semen Cells
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Semen cells were first stained with fluorescent antibodies such as CD3-APC-Cy7 (BioLegend, USA, clone SK7), CD4-BV605(BD, USA, clone RPA-T4), CD8-Percp/Cyanine5.5 (BioLegend, USA, clone SK1), CD56-BV421 (BioLegend, USA, clone HCD56), CCR5-FITC (BioLegend, USA, clone J418F1), CXCR4-APC (BioLegend, USA, clone RG5), p24-PE (Beckman Coulter, USA, clone KC57), and then analyzed using imaging flow cytometry.
The initial test of the staining plate contained all but one staining agent and fluorescence minus one control to determine the background staining of the channel. Cells were then acquired on an Amnis ImageStream Mk II flow cytometer (Luminex) using the INSPIRE 4.1 software with lasers set to maximum values without saturation in the brightest stains. Cell files (50,000) were collected with a cell classifier applied to the brightfield channel to capture a single-cell picture. Channels were as follows: Brightfield-Channel 1, FITC-Channel 2, PE-Channel 3, Percp/Cyanine5.5-Channel 5, BV421-Channel 7, BV605-Channel 10, APC-Channel 11, and APC/Cy7-Channel 12. Excitation lasers were used with the typical intensity settings of 405 nm (80 mW), 488 nm (100 mW), 594 nm (20 mW), and 658 nm (40 mW). All cell images were captured with the 40× objective and acquired at a rate of 200∼250 images per second. Data were analyzed using the IDEAS 6.2 (Amnis/EMDmillipore) software.
The initial test of the staining plate contained all but one staining agent and fluorescence minus one control to determine the background staining of the channel. Cells were then acquired on an Amnis ImageStream Mk II flow cytometer (Luminex) using the INSPIRE 4.1 software with lasers set to maximum values without saturation in the brightest stains. Cell files (50,000) were collected with a cell classifier applied to the brightfield channel to capture a single-cell picture. Channels were as follows: Brightfield-Channel 1, FITC-Channel 2, PE-Channel 3, Percp/Cyanine5.5-Channel 5, BV421-Channel 7, BV605-Channel 10, APC-Channel 11, and APC/Cy7-Channel 12. Excitation lasers were used with the typical intensity settings of 405 nm (80 mW), 488 nm (100 mW), 594 nm (20 mW), and 658 nm (40 mW). All cell images were captured with the 40× objective and acquired at a rate of 200∼250 images per second. Data were analyzed using the IDEAS 6.2 (Amnis/EMDmillipore) software.
6
Multiparameter Immune Cell Analysis
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CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, CD27-PE-Cy7, CD19-PE-TxR, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).
7
PBMC immunophenotyping and activation
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PMA/ionomycin-stimulated PBMCs were stained using fixable AQUA live/dead stain (Invitrogen) and the following antibodies: CD3-BUV395 (Invitrogen), CD4-BV605 (BD Biosciences), CD45RA-PerCPVio700 (BD Biosciences), CD31-BV421 (BD Biosciences), CD45RO-PE (BD Biosciences), and CD8-APC-Cy7 (BD Biosciences) in FACS buffer (phosphate-buffered saline containing 0.2% bovine serum albumin and 0.02% sodium azide). PBMCs were fixed and permeabilized (Foxp3, eBioscience). The PBMCs were intracellular stained with Ki67-AF647 (BD Pharmigen, measured in un-stimulated PBMCs) and IL-8-AF488 (CXCL8, BD Biosciences, measured in stimulated PBMCs) and incubated in the dark on ice for 1 hour. Samples were analyzed on an LSRII (Becton Dickinson) using FACSDiva software v.8.0. Subsequent data analysis was performed using FlowJo software 10.4.
8
Multiparameter Flow Cytometry Profiling of Activated cTfh
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CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, TIGIT-PE-Cy7, CD45RO-PECF594, CCR7-BV421, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).
Cells were washed twice and run/sorted on a BD FACS Aria III. Samples from 12 study participants were selected based on the magnitude of their cTfh response from d0 to d7 to undergo bulk sorting, with samples from four of these participants also undergoing single cell sorting. Sorted cells had 100U/mL of RNaseOUT added prior to sorting. Bulk sorted cells were centrifuged with the supernatant removed prior to freezing at -80°C. Data were analysed with FlowJo 10.1 (TreeStar). Activated and resting cTfh cells (CD4+CXCR5+PD-1+) were defined as CD38+ICOS+ and CD38-ICOS-, respectively (19 (link), 22 (link)). Activated plasmablasts were defined as CD19+CD27+CD38+. An example of flow gating for activation and sorting is depicted inSupplementary Figure 8
Cells were washed twice and run/sorted on a BD FACS Aria III. Samples from 12 study participants were selected based on the magnitude of their cTfh response from d0 to d7 to undergo bulk sorting, with samples from four of these participants also undergoing single cell sorting. Sorted cells had 100U/mL of RNaseOUT added prior to sorting. Bulk sorted cells were centrifuged with the supernatant removed prior to freezing at -80°C. Data were analysed with FlowJo 10.1 (TreeStar). Activated and resting cTfh cells (CD4+CXCR5+PD-1+) were defined as CD38+ICOS+ and CD38-ICOS-, respectively (19 (link), 22 (link)). Activated plasmablasts were defined as CD19+CD27+CD38+. An example of flow gating for activation and sorting is depicted in
9
Detailed Immunophenotyping of T-cell Subsets
10
Multiparametric Flow Cytometry Analysis
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Cell phenotypes were evaluated by multiparameter flow cytometry. Cells were incubated with a panel of labeled monoclonal antibodies (mAbs) anti-CD3 AF700, CD4 BV605, CD8 APC-H7, CD25 BV786, PD-1 APC, TIGIT BV421, and LAG3 BV711 (BD Biosciences) in staining buffer on ice in the dark for 20 min. Cells were then washed in the staining buffer and re-suspended in staining buffer and analyzed on an LSRII flow cytometer. Isotype controls were used for each experiment. Analysis of the results used FlowJo 10 software.
CFSE staining was conducted according to manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cells were labeled with CFSE by adding 1 mL of freshly prepared CFSE (2 μM in PBS containing 2% EV free FCS) to cells (up to 1×108 cells) in 1 mL of PBS 2% EV free FCS. The tube containing this mixture was covered with foil and incubated at 337°Cfor 5 minutes. Cells were pelleted, washed twice with 10 mL of PBS 2% EV free FCS, resuspended, counted and used for experiments.
CFSE staining was conducted according to manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cells were labeled with CFSE by adding 1 mL of freshly prepared CFSE (2 μM in PBS containing 2% EV free FCS) to cells (up to 1×108 cells) in 1 mL of PBS 2% EV free FCS. The tube containing this mixture was covered with foil and incubated at 337°Cfor 5 minutes. Cells were pelleted, washed twice with 10 mL of PBS 2% EV free FCS, resuspended, counted and used for experiments.
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