PBMC were isolated from patient blood by Ficoll-Paque (GE), and cryopreserved and stored in liquid nitrogen before analysis. PBMC were thawed, and 5 x 105 cells were stained for 30 min at 4° C with the following antibodies: CD3-APC-Cy7 (OKT3, Biolegend), CD4-PerCP-Cy5.5 (OKT4, Biolegend), CD8-APC (SK1, Biolegend), CD38-BV421 (HIT2, Biolegend), CD69-BV510 (FN50, Biolegend), HLADR-FITC (L243, Biolegend), CD103-PE (BerACT8, Biolegend), CCR5-PE-Cy7 (J418F2, Biolegend). Cells were then washed in FACS buffer and fixed in 1% paraformaldehyde before being acquired on a BD FACS Canto II. 1 x 106 PBMC were stimulated with a PMA/ionomycin cell stimulation cocktail (eBioscience), and treated with a protein transport inhibitor cocktail (eBioscience) for 4 h at 37° C, 5% CO2. Cells were then stained for 30 min at 4° C with the following antibodies: CD3-APC-Cy7 (OKT3, Biolegend), CD4-PerCP-Cy5.5 (OKT4, Biolegend), CD8-APC (SK1, Biolegend). Cells were washed and treated with fix/perm buffer (eBioscience) for 30 min at 4° C, before being stained for intracellular cytokines for 30 min at 4° C with the following antibodies: IFN-γ-PE (4SB3, Biolegend), TNFα-APC-Cy7 (MAb11, Biolegend). Cells were then washed in FACS buffer and fixed in 1% paraformaldehyde before being acquired on a BD FACS Canto II.
Cd3 apc cy7
Manufactured by BioLegend
Sourced in United States, Germany, Japan
CD3-APC-Cy7 is a fluorescently labeled antibody that binds to the CD3 antigen, which is expressed on the surface of T cells. It combines the APC and Cy7 fluorophores.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 fitc viobright, by Miltenyi Biotec (9 mentions)
Cd8 pe cy7, by BioLegend (9 mentions)
Cd4 apc, by BioLegend (8 mentions)
Canto 2, by BD (8 mentions)
Cd19 apc cy7, by BioLegend (7 mentions)
Ly6g pe cy7, by BioLegend (5 mentions)
Cd38 pe cy7, by BioLegend (5 mentions)
Cd45 percp cy5, by BioLegend (4 mentions)
Cd64 apc, by BioLegend (4 mentions)
Cd45 af700, by BioLegend (4 mentions)
Cd19 pe, by BioLegend (4 mentions)
Cd8a fitc, by BioLegend (4 mentions)
Cd14 apc cy7, by BD (4 mentions)
Cd21 apc, by BioLegend (4 mentions)
Cd27 bv650, by BioLegend (4 mentions)
Cd4 pe cy7, by BioLegend (4 mentions)
Facsaria 2, by BD (4 mentions)
Cd11b bv510, by BioLegend (3 mentions)
Cd11b apc, by BioLegend (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
102 protocols using cd3 apc cy7
1
Phenotypic and Functional Analysis of PBMC
2
Multiparametric Imaging Flow Cytometry of Semen Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
3
Characterizing Cardiac Immune Populations
4
Multiparameter Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue 450, CD8-FITC (Tonbo), CD3-viogreen (Miltenyi), CD45-AF700, CD3-APC-Cy7, CD4-APC-Cy7, CD103–BV711 (Biolegend), CD4-PE-Cy5.5, CD103–PE-Cy7 (eBioscience, San Diego, CA), CD8-BUV395 (BD Bioscience). Analysis was performed on BioRad ZE5 flow cytometers (BioRad) using Everest software or Gallios (Beckman Coulter) using Kaluza software, and data analyzed with FlowJo software (Tree Star, Inc. Ashland, OR). Expression of surface markers was measured by the percentage of positive cells.
5
Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed on a Fortessa (BD Biosciences) as previously described methods [2 ]. Anti-mouse CD19-Percp, CD8-PE-Cy7, F4/80-APC, CD3-APC-Cy7, CD11c-PE, CD4-FITC, and CD49b-PE were purchased from Biolegend.
6
T Cell Phenotyping and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein expression analysis following proliferation, CD8 or CD4 T cells were isolated and stimulated as described above. On day 4 of the proliferation assay cells were collected and stained with the following antibodies for surface protein expression: CD3 APC-Cy7 (Biolegend), CD4 AF-700 (Biolegend), CD8 PE-Cy7 (Biolegend), CD45RA BV-605 (Biolegend), CCR7 AF-488 (Biolegend) or CCR7 PE-Dazzle594 (Biolegend), and CD19 PerCP-Cy5.5 (Biolegend). Dead cells were excluded from the analysis by using Zombie-UV (Biolegend). Doublets and double positive CD4/CD8 cells were removed through sequential gating. Flow cytometry acquisition was done using the BD LSRII with BD FACSDiva and Cyteck Aurora. Data was analyzed by FlowJo 10.1r7 and GraphPad Prism 9.
7
Evaluating Dexamethasone's Impact on CTL Viability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Annexin V apoptosis assay was performed to evaluate the effect of dexamethasone on the viability of CTLs from Cas9 control and NR3C1 KO groups. CTLs from both groups were treated with 200 μM dexamethasone (Sigma) for 72 hours. Cells were then collected, washed with Annexin V buffer, and stained with Annexin V (V500; BD Biosciences) and live/dead viability dye (efluor 660; Invitrogen) in addition to CD3 APC Cy7 (Biolegend, Clone HIT3A), CD4 APC (E Biosciences, Clone SK3), and CD8 PerCP Cy5.5 (Biolegend, Clone SK1). The proportion of apoptotic (positive for Annexin V) and dead CTLs (positive for live/dead stain) was determined by flow cytometry.
To confirm the ability of SARS-CoV-2 CTLs to mediate cytotoxicity, autologous PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) per manufacturer’s recommendation and loaded with SARS-CoV-2 Spike (S) (peptide pool 1 or 2), Membrane (M) and Nucleocapsid (N) PepMix (JPT, Germany) [1μg /ml per peptide] overnight. The next day, the SARS-CoV-2 PepMix-loaded PBMCs were co-cultured with the expanded CTLs at 1:1 ratio for 16 hours followed by Annexin V staining as described above. The proportion of dead/apoptotic PBMCs (Live/Dead+ and Annexin V+) was determined by flow cyometry.
To confirm the ability of SARS-CoV-2 CTLs to mediate cytotoxicity, autologous PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) per manufacturer’s recommendation and loaded with SARS-CoV-2 Spike (S) (peptide pool 1 or 2), Membrane (M) and Nucleocapsid (N) PepMix (JPT, Germany) [1μg /ml per peptide] overnight. The next day, the SARS-CoV-2 PepMix-loaded PBMCs were co-cultured with the expanded CTLs at 1:1 ratio for 16 hours followed by Annexin V staining as described above. The proportion of dead/apoptotic PBMCs (Live/Dead+ and Annexin V+) was determined by flow cyometry.
8
Multiparametric Flow Cytometry of Murine Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 1 × 106 lymphocytes were acquired and collected from mice spleen, and then 0.5 μg anti-mouse CD16/32 monoclonal antibody (Biolegend, USA) and 5 μL rat serum were used to block the Fc receptor for 10 min. For measuring the number of total CD4+ T cell and naïve CD4+ T cell, splenocytes were incubated with 1 μg CD3-APC-Cy7 (Biolegend), CD4-APC (eBioscience, Thermo Fisher Scientific, USA), TCRβ-PE and CD62L-FITC (eBioscience, Thermo) for 30 min. For detecting cell activation, cells were stained with 1 μg CD25-PE and CD69-FITC (eBioscience, Thermo). For evaluating cell surface protein expression, EGFR (Thermo)-PE (eBioscience) and Glut1 (Proteintech, China) -FITC (eBioscience) were incubated for 30 min. To measure cell death, splenic cells were stained with PI and Annexin V (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature. Cells were analyzed by FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA). All data were analyzed by Flowjo10 software (Tree Star, Ashland, OR, USA).
9
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FACS analysis of cell surface CAR and protein expression was performed using a CytoFLEX S flow cytometer (Beckmam Coulter, CA, USA). CD22-CAR was detected by incubation with APC-F(ab)2 (Jackson Immunoresearch, USA). The following human monoclonal antibodies were used for detection of cell surface proteins: CD22-APC, CD22-PE, CD19-PE, CD45-PerCP/Cy5.5, CD3-APC/Cy7, CD8-Pacific Blue, CD4-APC, CD45RO-PE/Cy7, CD45RA-FITC, CCR7-APC (all from BioLegend). CD22 site density was quantified by Quantity-PE beads (BD Biosciences, USA) following instructions. Both GFP-expressing cells and CFSE dye-labeled cells were identified through the FITC channel.
10
Quantifying NK Cell Markers in Whole Blood and Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood and intratumoural immune cells were stained with CD56-FITC-Viobright, NKG2A-APC, CCR5-FITC, CCR1-APC, CCR3-PE (Miltenyi Biotec), CD3-APC-Cy7, NKp30-BV421, NKp46-PE-Cy7, NKG2D-PE-Cy5, PD-1-PE-Cy7, TIGIT-PE-Cy5, CD69-BV510 (BioLegend). Red blood cells were lysed using BD Lysing Solution (BD Biosciences) as per manufacturer’s instructions. NK cells were quantified as CD56+CD3− cells within the lymphocyte gate. Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (Tree Star).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!