Single cell suspensions were stained with three separate antibody cocktails for analyses of BM, spleen, and thymus. BM subsets were identified using the following conjugated mAbs: Ter119, CD25 (Invitrogen, Thermo Fisher Scientific, Waltham, MA), c-kit, Sca1, CD19 and CD44 (BD Biosciences, San Jose, CA). Lineage-differentiated BM cells were eliminated using a dump consisting of mAbs against CD11b, Gr1 and CD3. HSCs, MPPs, and HPCs were identified within the lineage- Sca1+c-kit+ (LSK) lineage as a function of CD150 and CD48 profiles while CMP, GMP, and MEP were evaluated on lineage-c-kit+ (LK) progenitors as a function of CD34 and CD16/32 expression (36 (link), 38 (link)). For thymus, cells were stained with the following directly conjugated mAbs: CD3, c-kit, CD4, CD8, CD44, TCRγδ (BD Biosciences) and CD25 (Invitrogen). Non-T lineage cells were excluded using a dump consisting of mAbs against CD19, Gr1, CD11b. Splenic subsets were distinguished using the following directly conjugated mAbs: Ter119 (Invitrogen), CD44, CD3, CD4, CD8, CD62L, TCRγδ, CD19 and CD11b (BD Biosciences). Stained cells were analyzed by flow cytometry using the LSR II-Fortessa (BD Biosciences). All data analyses were performed using Diva (BD Biosciences), and FlowJo Mac v.10.6.2 software (Tree Star).
Tcrγδ
Manufactured by BD
Sourced in United States
TCRγδ is a type of T cell receptor expressed on the surface of a subset of T cells. It plays a role in the recognition of specific antigens by the immune system.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BD (4 mentions)
Cd62l, by BD (4 mentions)
Facscanto 2, by BD (4 mentions)
C kit, by BD (3 mentions)
Cd11c, by BD (3 mentions)
Fluorescein isothiocyanate fitc conjugated, by BD (2 mentions)
Tcrαβ, by BD (2 mentions)
Ter119, by BD (2 mentions)
Cd45ra, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Tcrαβ, by BioLegend (2 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
Lsrii fortessa, by BD (1 mentions)
Mac 1, by BD (1 mentions)
Igm fitc, by Thermo Fisher Scientific (1 mentions)
Facslyric flow cytometer, by BD (1 mentions)
Tcr vβ repertoire kit, by Beckman Coulter (1 mentions)
Propidium iodide, by BioLegend (1 mentions)
α cd8, by BioLegend (1 mentions)
α cd4, by BioLegend (1 mentions)
17 protocols using tcrγδ
1
Multiparametric Flow Cytometric Analysis of Hematopoietic Subsets
2
Multicolor Flow Cytometry Antibody Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were purchased from BD Biosciences: fluorescein isothiocyanate (FITC)-conjugated erythroid lineage cells (TER119; 561032), MAC1 (553310), GR1 (553127), CD11C (557400), B220 (553088), THY1.2 (553004), CD8A (553031), CD4 (553651), NK1.1 (553164), CD3ε (553062), CD19 (553785), TCRγδ (553177), phycoerythrin (PE)-conjugated SCA-1 (553336), CD4 (553653), CD19 (553786), GR-1 (553128), NK1.1 (553165), TCRβ (553172), allophycocyanin (APC)-conjugated LY5.1 (558701), LY5.2 (558702), C-KIT (553556), and CD19 (550992). FITC-IGM (11-5790-81) was purchased from eBioscience.
3
Comprehensive T-cell and NK-cell Phenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T-cell and NK-cell flow cytometry was performed as previously described [13 (link), 23 (link), 24 (link)], on a FACSCanto II or FACSLyric flow cytometer (BD Biosciences, San Jose, CA), with antibodies to CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A, TCR γ/δ (BD Biosciences, San Jose, CA), and TRBC1 (clone JOVI-1; Ancell Corp, Bayport, MN). The TCR Vβ repertoire kit (Beckman Coulter, Indianapolis, IN) was used to detect TCR Vβ clonality.
4
Isolation and Analysis of Cytotoxic T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells were enriched from PBMCs by negative selection using magnetic bead separation according to the manufacturer’s instructions (STEMCELL Technologies), added to uninfected or infected A549 cells at a ratio of 3:1, and incubated for 16 h in the presence of α-TNF (Beckman Coulter) and TAPI-O (EMD Millipore) to prevent TNF cleavage from the cell surface (Haney et al., 2011 (link)). The following day, cells were stained with: (a) α-CD4 and α-CD8 (BioLegend) and (b) α–TRAV1-2 and α–TCRγδ (Thermo Fisher Scientific). Dead cells were excluded from the analysis using propidium iodide (BioLegend). Viable CD4−TCRγδ−CD8+TRAV1-2+TNF+ cells were sorted at high purity using a FACSAria flow cytometer (BD) directly into RNAlater (Applied Biosystems). The corresponding nonfunctional populations were sorted in parallel for control purposes. In all cases, cell numbers were standardized (5,000 per aliquot) to minimize sampling bias.
5
Lymphocyte Immunophenotyping in Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood was collected from study patients in evacuated tubes containing sodium heparin anticoagulant at 4 weekly time points. Blood was obtained once from healthy volunteers and disease controls. Single platform flow cytometric lymphocyte immunophenotyping was performed for each specimen using whole blood lysis technique after labeling with fluorochrome-conjugated monoclonal antibodies to CD3, CD4, CD8, CD16/56, CD19, CD27, CD28, CD45, CD45RA, CD25, CD57, CD127, CD197, and TCR γδ (BD Biosciences, San Jose, CA). The correlative biologic studies associated with this trial were designed for simple phenotyping in a setting that would be adaptable to a larger trial had the results been more favorable. In keeping with design, surface phenotyping was preferred if specimens were to be analyzed fresh at multiple centers. The BD Treg kit was specifically validated for the negative correlation of FOXP3 and CD127 (IL-7rα) expression and CD127 is a validated marker for this purpose as described by Simonetta.[8 (link)] Specimens were acquired in TruCount polystyrene tubes on a 3-laser 8-color FACS Canto II flow cytometer, stored as list mode files, and analyzed using FACS Canto and DiVa software (BD Biosciences, San Jose, CA).
6
Tumor Immune Microenvironment Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor specimens with adjacent tissues, relevant internal organs and inguinal sentinel lymph nodes (SLN) were harvested and fixed in 4% neutral buffered formalin solution for paraffin sectioning and staining. Immunohistochemical test for in situ detection were performed with anti-MUC-1 (BD), CD49b/NK1.1 (Biolegend), anti-CD4, CD8, CD25, Foxp3, β-catenin, TERT (all abcam), and anti-CD44, CD133 (all Chemicon) monoclonal antibody severally. Imaging of thymic lobules for renewal hotspots were captured using Leica scanning confocal microscope. Thymic and tumor tissue/cells were stained with thymosin-α1(abcam)/-β4(Millipore) for immunofluorescence or with TCRαβ, TCRγδ, CD3, CD38, CD80, CD163, CD45RA (all BD) for positive subsets via FACS-Aria III cell sorting system. Successive subsets were incubated with 3D-CSC-subsets from in vivo tumor at a ratio of 120:1 (thymocyte: spheroid) in DMEM/F12 with 2% normal serum for over 72 hours of reactivity between renovated T-subsets and CSC pool.
7
T-cell Immunophenotyping Protocol
8
Isolation and Sorting of CD4 Memory Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood cells were obtained by leukapheresis. Following lysis of red blood cells, they were preserved in freezing medium at -80C/LN. 3.0 × 108 PBMC (93.1% viability) were thawed and stained with LIVE/DEAD Aqua stain (Molecular Probes, L34957, 5 min, 22°C), CD3-APC-H7 (BD Biosciences, 641406), CD4-BV785 (BioLegend, 317442), CD8-QDot655 (Invitrogen, Q10055), CD11c-PE (BD Biosciences, 347637), CD14-PE (BD Biosciences, 555398), CD27-PE/Cy5 (Beckman Coulter, 6607107), CD45RO-ECD (Beckman Coulter, IM2712U), CD56-APC (BioLegend, 304610), CD57-BV421 (VRC Ab), CCR7-Ax700 (VRC Ab), and TCR γδ (BD Biosciences, 555718) for 15 min at 22°C. Cells were washed, kept on ice, and sorted on a BD FACSAria into CD4 memory subsets as follows: Naïve (CD3+ Aqua-CD8-CD4hiCD56-TCRγδ-CD14-CD11c-CD27 + CD45RO-CCR7 + CD57-), Central/Transitional Memory (CTM, CD3+ Aqua-CD8-CD4hiCD56-TCRγδ-CD14-CD11c-CD27+ CD45RO+), and Effector Memory (EM, CD3 + Aqua-CD8-CD4hiCD56-TCRγδ-CD14-CD11c-CD27-) (Supplementary Figure S1 ).
9
Comprehensive Mouse Immune Cell Profiling
10
Isolation and Sorting of Innate Lymphoid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For further purification, MNCs from freshly resected patient tissue specimens were subjected to Ficoll‐Hypaque gradient centrifugation for 30 min at 24°C. Next, the MNC layer was transferred to a new tube, washed twice with phosphate-buffered saline (PBS), and suspended in PBS. ILCs were sorted using a BD FACSAria system (BD Bioscience) as Lin−-enriched MNCs as Lin cocktail− (CD3, CD19, CD20, and CD14), CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ CD127+ CRTH2+/− CD117+/− cells using FITC anti-Lin (643510; BD Bioscience), CD94 (305504; Biolegend, San Diego, CA, USA), CD34 (343504; Biolegend), CD1a (300104; Biolegend), T cell receptor (TCR)α/β (306706; Biolegend), TCRγ/δ (331208; Biolegend), allophycocyanin (APC)-H7 anti-CD45 (56017; Biolegend), Percp-cy5.5 anti-CD127 (351322; Biolegend), phycoerythrin (PE)-Cy7 anti-CRTH2 (350118; Biolegend), and BV605 anti-CD117 (562687; Biolegend). pDCs were sorted as Lin− CD94− CD34− CD1a− TCRα/β− TCRγ/δ− CD45+ BDCA2+ cells using FITC anti-Lin, CD94, CD34, CD1a, TCRα/β, TCRγ/δ, APC-H7 anti-CD45, and APC anti-BDCA2 (17-9818-42; Biolegend). Purity was routinely >99%. Cell viability was determined by trypan blue staining and was >99% after isolation.
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!