Tumour and liver samples were enzymatically digested (0.25 mg/mL liberase (Sigma) and 1 mg/mL DNAse (Sigma) in DMEM) at 37 °C for 40 min. Cell suspensions were filtered (100 μm cell filters, Corning), treated with red blood cell lysis buffer for 2 min at 37 °C, washed, and resuspended at 1 × 107 cells/mL in cold FACS wash buffer (10% bovine serum albumin/5 mM EDTA/0.01% sodium azide in PBS pH 7). Briefly, 1 × 106 cell aliquots were stained with either Cocktail 1: CD45-BV510, CD4-FITC, CD3-PE, CD44-PerCP, CD8-PECy7, PD-1-APC, CD69-APC-Cy7 (BD, Biosciences) and viability dye DAPI, Cocktail 2: CD45-BV510, CD103-FITC, CD3-PE, CD8-PECy7, PD-1-APC, CD4-APC-Cy7 (BD, Biosciences) and DAPI, or Cocktail 3: CD45-BV510, F4/80-FITC, Ly6C-PE, PD-L1-PECy7, CD11b-APC-Cy7 (BD, Biosciences), and DAPI. Samples were run on the Canto II (BD Biosciences) and analysed using FlowJo™ software.
Pd 1 apc
Manufactured by BD
Sourced in United States
PD-1-APC is a flow cytometry antibody that binds to the Programmed Cell Death 1 (PD-1) protein. PD-1 is an inhibitory receptor expressed on T cells and other immune cells. The PD-1-APC antibody is conjugated with the fluorescent dye Allophycocyanin (APC), allowing for the detection and analysis of PD-1-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by BD (3 mentions)
Cd3 apc cy7, by BD (2 mentions)
Tigit pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd28 pe cy7, by BD (2 mentions)
Cd127 pe cy7, by BD (2 mentions)
Fas pe, by BD (2 mentions)
Ctla 4 pe, by BD (2 mentions)
Pd l1 apc, by BD (2 mentions)
Cd8 pe cy7, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Lag3 pe, by BD (1 mentions)
Cd8 pe cy5, by BD (1 mentions)
Fluorescent microspheres, by Bangs Laboratories (1 mentions)
Annexin 5 fitc pi kit, by BioLegend (1 mentions)
Flash red, by Bangs Laboratories (1 mentions)
Cd4 pe cf594, by BD (1 mentions)
Cd25 alexa 700, by BioLegend (1 mentions)
Tim 3 bv711, by BioLegend (1 mentions)
Ctla 4 percpcy5, by BioLegend (1 mentions)
Cd8 bv785, by BioLegend (1 mentions)
15 protocols using pd 1 apc
1
Multiparametric Flow Cytometry of Tumor and Liver Samples
2
Comprehensive Immunophenotyping Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All flow cytometry antibodies were purchased from BD Pharmingen (San Diego, CA, USA), BioLegend (San Diego, CA, USA) and KeyGen Biotech (Nanjin, Jiangsu, China). The following antibodies were obtained from BD Pharmingen™: CD3-APC-Cy™7, CD4-FITC, CD8-PECy™5, PD-1-APC, and LAG3-PE. The following reagents were obtained from BioLegend: PerCP/Cy5.5-conjugated anti-IL-2, PE/Cy7-conjugated anti-IL-6, PerCP/Cy5.5-conjugated anti-TNF-α, and PE/Cy7-conjugated anti-IFN-γ antibodies, a FITC Annexin V/PI kit, and a KGA: FITC-BrdU kit. The following quantum MESF beads were purchased from Bangs Laboratories: Fluorescent Microspheres, Intensity Standard: Dragon Green, Flash Red, PE-MESF, and APC-MESF. The PMA/ionomycin mixture (250X) was purchased from MultiSciences (Lianke) Biotech (Hangzhou, Zhejiang, China). Enzyme-linked immuno sorbent assay (ELISA) kits for human interleukin-2 (IL-2), IL-6, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were purchased from Beijing 4A Biotech (Beijing, China). Brefeldin A was purchased from Qcbio Science & Technologies Co., Ltd. (Beijing, China).
3
T Cell Inhibitory Receptor Profiling in RA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells from HCs and paired PBMCs and SFMCs from RA patients were stained for the presence of T cell co-inhibitory receptors. Non-specific binding was blocked by 50 µg/ml mouse IgG (Jackson), and surface staining was performed using the following antibodies: CD3 V450 (clone: UCHT1, BD), CD4 PE-CF594 (clone: RPA-T4, BD), CD8 BV785 (clone: RPA-T8, BioLegend), CD25 Alexa 700 (clone: BC96, BioLegend), Tim-3 BV711 (clone: F38-2E2, BioLegend), CTLA-4 PerCPCy5.5 (clone: L3D10, BioLegend), Tigit PE-Cy7 (clone: MBSA43, eBioscience), PD-1 APC (clone: MIH4, BD), Live-dead near IR (Thermo Fisher Scientific). Cells were permeabilized by BD Facs lysing solution and BD Facs Perm Solution 2 (both BD bioscience). Intracellular staining was performed using Eomes PE (clone: WD1928 ebioscience). All antibodies were used in the concentration recommended by the manufacturer. Gating was done on lymphocytes, excluding doublets and dead cells. Gates were set using FMOs. CD3+ CD4+ and CD3+ CD8+ cells were investigated for their expression of T cell co-inhibitory receptors.
4
Comprehensive Immune Cell Profiling Post-Transplantation
5
Comprehensive CD4+ T Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One day post isolation FACS analysis was used to determine the percentages of various CD4+ T cell subsets, like naïve (TN), effector memory (TEM), central memory (TCM), T helper 17 cells (Th17), follicular helper T cells (Tfh) and regulatory T cells (Treg). For this the following antibodies were used: CD4-APC H7 (BD Biosciences, 560158), CD45RA-PE Cy7 (BD Biosciences, 560675), CD45RO-PerCP Cy5.5 (BD Biosciences, 560607), CCR7-APC (BioLegend, 353214), CCR6-PE (BD Biosciences, 559562), CXCR5-PerCP Cy5.5 (BioLegend, 335001), CD127-PE Cy7 (BD Biosciences, 560822), CD25-Horizon V450 (BD Biosciences, 560355). Further, the expression of different receptors on unstimulated as well as stimulated CD4+ T cells was determined using the following antibodies: CD4-APC H7, TCR-CD3-APC H7 (BD Biosciences, 560275), CD28-PerCP Cy5.5 (BD Biosciences, 560685), MHC-I-Horizon V450 (BD Biosciences, 561346), FAS-PE (BD Biosciences, 556641), FAS-L-PE (BioLegend, 306407), PD1-APC (BD Biosciences, 558694), PD1-L-PE Cy7 (BD Biosciences, 558017), TRAIL-PE (BD Biosciences, 550516), CTLA-4-PE (BD Biosciences, 555853), CD69-Horizon V450 (BD Biosciences, 560740), CD25-PE Cy7 (BD Biosciences, 557741), CXCR4-PerCP Cy5.5 (BD Biosciences, 560670) and CCR5-PE Cy7 (BD Biosciences, 557752). Cells were analyzed using the BD FACS Canto II with FACSDiva software.
6
Comprehensive NK cell phenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs from subjects were resuspended in PBS buffer and were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were washed with 1× PBS before flow cytometry analysis. Antibodies used included anti-human CD3-BV786, CD19-BVAPC-H7, CD16-BV711, CD69 PE-CF594, CD57-BV421, PD-1-APC, CD70-PE, CD160-AF488, 2B4-AF700, CTLA-4-PE, TIM-3-FITC, 7-AAD (BD Biosciences, San Diego, CA, USA), CD56-BV510, BTLA-PE-CY7, CD39-APC (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7 (Ebioscience, San Diego, CA, USA), along with the corresponding isotype controls. NK cells were gated as CD3−CD14−CD19−CD56+CD16+, CD3−CD14−CD19−CD56+CD16−, or CD3−CD14−CD19−CD56−CD16+. Data were acquired with the LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.5 (Tree Star, Ashland, OR, USA).
7
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
8
Profiling PD-1 and LAG-3 expression on T cells
9
Comprehensive Immunophenotyping of Lung Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry was performed using fluorescence-conjugates or specific mAb and their controls followed by species-specific conjugate (Supplementary Table 2 ) using a FACS CantoII flow cytometer (Beckman Coulter) or a LSRFortessa (Becton Dickinson) from the flow cytometer facility Tuebingen. Positive cells in percentage (%) were calculated as follows: Surface expression in percent obtained with the specific antibody—surface expression in percent obtained with isotype control. Platelets were preselected by CD41a+ and CD62P− (resting) or CD62P+ (activated). Data analysis was performed using FlowJo software (v.10). In order to verify the reproducibility of our flow cytometry system, we performed a Bland–Altman analysis (Supplementary Fig. 8f ). For immunophenotyping of PBMC subsets of lung cancer patients and healthy control donors were identified by counterstaining with CD3-PECy5 (BD biosciences, San Diego, CA), CD19-APC/Fire750, CD4-Pacific Blue, CD8a-BV605, CD56-PECy7, CD14-BV785, HLA-DR-BV650 (Biolegend, San Diego, CA) and CD16-FITC (invitrogen). PD-1 and PDL-1 expression as well as activation levels were analyzed using a PD-1-APC or PDL-1-APC and a CD69-PE antibody (BD biosciences), respectively. Isotype controls were obtained from BD biosciences. Dead cells were excluded from analysis with LIVE/DEAD™ Fixable Aqua (Thermo Fisher Scientific, Waltham, MA).
10
Flow Cytometry Analysis of PBMC Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometry, whole-blood specimens were processed within 4 hours of collection to isolate PBMCs, using Lymphoprep (Axis-Shields-Diagnostics) as previously described [27 (link)]. Cells were analyzed using a CyAn ADP 9 color flow cytometer (Beckman Coulter). The T-cell panel included CD3 BV510, CD4 V450, CD38 PE Cy7, HLA-DR AF700, PD-1 APC, and CD57 FITC (all from BD Biosciences) and CD8 PE (Biolegend). The monocyte panel included HLA-DR AF700, CD14 PE Cy7, and CD16 PE (all from BD Biosciences). Anti-mouse Igk isotype control and negative control particles (BD Biosciences) were used for compensation. A standardized gating strategy was followed for T cells (Supplementary Figure 1A ) and monocytes (Supplementary Figure 1B ). Monocyte subsets were identified as previously described [28 (link)].
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!