Single cell suspensions were prepared from the lungs, draining lymph nodes and spleen tissues collected 1 week after the booster immunization. Activated T cells in the lung were identified using anti-CD44 antibody. HA-specific CD8+ T cells in the lungs, lymph nodes and spleens were identified using H-2Kd/IYSTVASSL tetramer (NIH Tetramer Core Facility, Emory University, Atlanta, GA, USA). To detect intracellular cytokine production by CD4 or CD8 T cells, 106 cells from the spleen were stimulated in vitro with RG A/IN/05 virus at an MOI of 1 for 16 h or with HA peptide (1 µM, NeoBiolab, Woburn, MA, USA) for 6 h, respectively. GolgiPlug™ (BD Bioscience, San Jose, CA, USA) was added during the last 5 h of incubation. Cells were surface stained with either anti-CD4 or anti-CD8 antibody (BD Bioscience), followed by intracellular staining with anti-IFN-γ antibody (BD Bioscience). Samples were analyzed using an LSRII Flow cytometer (BD Biosciences), and the cytometric data were analyzed using FlowJo software version 9.3.3 (Tree Star, Inc., Ashland, OR, USA).
Anti cd8 antibody
Manufactured by BD
Sourced in United States
The Anti-CD8 antibody is a laboratory reagent used in flow cytometry and other immunological applications to identify and analyze CD8-positive cells, such as cytotoxic T cells. The antibody specifically binds to the CD8 surface marker expressed on a subset of T lymphocytes.
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Lab products found in correlation
Anti cd45 antibody, by BD (3 mentions)
Anti ifn γ antibody, by BD (2 mentions)
Anti cd3 antibody, by BD (2 mentions)
Anti cd4 antibody, by BD (2 mentions)
Anti cd11b antibody, by BD (2 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Anti cd4, by BD (1 mentions)
Fcs express version 5, by De Novo Software (1 mentions)
Anti 7 aad antibody, by BD (1 mentions)
Anti cd56 antibody, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Anti cd107a antibody, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Fixable viability stain 620, by BD (1 mentions)
Anti cd45ra antibody, by BD (1 mentions)
Anti pd 1 antibody, by BD (1 mentions)
Folate, by Merck Group (1 mentions)
Anti cd11c antibody, by BD (1 mentions)
11 protocols using anti cd8 antibody
1
Quantifying influenza-specific T cell responses
2
Immunophenotyping of Expanded Cell Populations
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In order to analyze the cellular composition of the cell populations before and after expansion, the cells were stained using an anti-CD3 antibody (Clone: UCHT1; BD Biosciences, Franklin Lakes, NJ, USA) in combination with either an anti-CD56 antibody (Clone: NCAM16.2; BD Biosciences) or an anti-CD8 antibody (Clone: SK1; BD Biosciences). Isotype-stained cells served as controls. Additionally, all the cells were stained with an anti-7-AAD antibody (BD Biosciences) to exclude nonviable cells. Prior to staining, the cells were cryopreserved as previously described [19 (link)]. Immunofluorescence was measured via a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with CellQuest Pro software (BD Biosciences). The data were analyzed using FCS Express, version 5 (DeNovo Software, Glendale, CA, USA).
3
T2 Cell Line Maintenance and Antibody Production
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The American Type Culture Collection (ATCC, USA) provided the Human TAP-deficient T2 cell line, the BB7.2 cell line that produced mAb against HLA-A*0201, and the U251 cell line. All of the cell lines were maintained in RPMI 1640 with 10% FCS and 100 μg mL−1 of penicillin and streptomycin. Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FCS, 4 μg L−1 glucose, and 100 μg mL−1 penicillin/streptomycin was used to maintain the BB7.2 cell line. Every cell line was maintained in a humidified environment with 5% CO2 in the air at 37 °C. Anti-HLA-A2 antibody was produced using BB7.2 (106 T2 cells/100 μL hybridoma culture supernatant). We bought the anti-CD8 antibody from BD Biosciences Pharmingen in the United States. Transgenic (Tg) HLA-A*0201/Kb mice, aged 8–12 weeks, were acquired from The Jackson Laboratory (USA). Mice were raised and housed in facilities designated as pathogen-free (SPF). Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Chongqing Medical University.
4
Quantifying CSPG4-CAR T Cell Degranulation
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Degranulation of CSPG4-CAR T cells towards leukemia cells was measured using conventional CD107a staining. T cells, either mock-electroporated or transfected with a CEA-specific CAR, served as controls. Twenty-four hours after electroporation, T cells were stimulated at a 1:1 ratio with T2.A1, KOPN8, and A375M target cells. Monensin (eBioscience, San Diego, CA, USA) at a final concentration of 1 µM and an anti-CD107a antibody (BD Biosciences, San Jose, CA, USA, clone: H4A3) were added right at the beginning of co-culture. After 4 h, the cells were stained with an anti-CD8 antibody (BD Biosciences, San Jose, CA, USA, clone: SK1) and analyzed via flow cytometry. Degranulation of CD8-positive T cells was calculated by dividing the portion of CD107a-positive/CD8-positive cells by the portion of CD8-positive cells.
5
Multi-Marker Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
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Single-cell suspension obtained and stained by following mAbs: Live/dead dye (Fixable Viability stain 620, BD Biosciences, 564,996), anti-CD45 antibody (BD Biosciences, 560,178), anti-CD3 antibody (BD Biosciences, 563,800), anti-CD4 antibody (BD Biosciences, 566,392), anti-CD8 antibody (BD Biosciences, 563,821), anti-CD25 antibody (BD Biosciences, 564,467), anti-CD127 antibody (BD Biosciences, 558,598), anti-CD45RA antibody (BD Biosciences, 563,031), anti-CCR7 antibody (BD Biosciences, 562,555), anti-PD-1 antibody (BD Biosciences, 563,245), anti-LAG3 antibody (BD Biosciences, 565,774), anti-CD39 antibody (BD Biosciences, 561,444). Single cell suspensions were stained with 1 μg/sample fluorochrome-labeled antibodies for specific surface marker at 4°C for 30 min in 100 μL PBS. Stained single cell suspension of tumor tissue were processed to flow cytometry using Cytoflex LX. The data were analysis by using FlowJo V10.6.2 software.
6
Nanoparticle-based Immunostimulant Delivery
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Folate (FA), monomethoxyl poly (ethylene glycol) (MPEG), ε-CL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) were purchased from Sigma–Aldrich (Darmstadt, Germany). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Avanti Polar Lipids (AL, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were both purchased from Gibco (NY, USA). The antibodies used in this study were summarized: rat anti-mouse CD31 polyclonal antibody, rat anti-mouse Ki67 polyclonal antibody, rat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Servicebio, Wuhan, China); The anti-CD4 antibody, anti-CD8 antibody, anti-CD69 antibody, anti-CD11c antibody, anti-CD80 antibody, anti-CD86 antibody, anti-MHCII antiobody, anti-CD45 antibody, anti-CD11b antibody and anti-IFN-γ antibody (BD Pharmingen, NJ, USA); The anti-CD11b antibody, anti-CD206 antibody, anti-F480 antibody, anti-CD107a antibody, anti-CD49b antibody, anti-CD274 (PDL-1) antibody (Biolegend, CA, USA). The pvax and pIL-12 were extracted and purified using a QIAGEN Endofree Plasmid Mega Kit (Hulsterweg, Netherlands). The contents of murine IFN-γ, TNF-α and IL-12 p70 were detected by corresponding ELISA kits from Invitrogen (CA, USA).7-AAD/Annexin-V apoptosis kit was purchased from BD, Pharmingen (CA, USA).
7
Modulating T Cell Proliferation Using IL-1-activated Fibroblasts
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The influence of the IL−1-activated CAFs on T cell proliferation was assessed using a murine T cell proliferation assay. Briefly, 100,000 mouse fibroblasts (L-control cells) were treated with 1 ng/ml IL-1β in the presence of 100 ng/ml of anti-IL1R1 or 100 ng/ml of control IgG. After 24 h, cells were washed with PBS (1×, Gibco) and loaded with the LCMV gp33−41 peptide (1 µg/ml, RP20257, BioConnect) for 2 h. Gp33−41-specific CD8+ T cells were isolated from splenocytes of P14 TCR transgenic mice (provided by Prof. D. Brenner) using immune-magnetic separation and labeled with carboxyfluorescein succinimidyl ester (CFSE). Labeled CD8+ T cells were co-cultured with gp33-loaded fibroblast cells in presence of murine IL-2. After 72 h, T cells were stained with an anti-CD8 antibody (558106, BD Biosciences) and the proliferation was measured by flow cytometry as described above.
8
Identifying HLA Restriction in T-Cell Assays
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HLA-I-/II-restriction was determined by ELISpot assays using antibodies blocking either CD4 or CD8. The cells were incubated at a final concentration of 50 μg/mL of an anti-CD4 (OriGene Technologies, Rockville, Maryland, USA) or 625 ng/mL of an anti-CD8 antibody (BD Bioscience, San Jose, CA, USA) for one hour at 37 °C and 7% CO2. Then, peptides were added directly to the wells at a final concentration of 20 μg/mL. The restricting HLA-I allele was determined using HLA-matched allogeneic B-LCLs from HLA-I-typed blood donors. These target cells were incubated with the respective peptides for one hour and washed twice with 15 mL PBS. Peptide-sensitized target cells and target cells without peptides were then co-incubated with the T-cell lines in a γ-IFN ELISpot assay, using 2 × 105 target cells per well and 2 × 105 T-cells in duplicate.
9
Multiparametric IHC and Flow Cytometry
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For IHC staining, we used the following antibodies: anti-ACE2 antibody (ab108252), anti-TMPRSS2 antibody (ab242384) and anti-OMP antibody (ab183947) from Abcam.cn. Furthermore, ServiceBio Company provided the following antibodies: anti-CD19 antibody (GB11061-1); anti-CD45 antibody (GB11066); anti-F4/80 antibody (GB11027); anti-CD3 antibody (ab16669); anti-CD4 antibody (GB13064-2); anti-CD8 antibody (GB13429); anti-CD11b antibody (GB11058); and anti-CD11C antibody (GB11059).
For flow cytometry, we used the following antibodies: anti-TCRβ antibody (FITC, BD 553170); anti-TCRγδ antibody (PE, BD 553178); anti-CD8a antibody (PP5.5, BD 551162); anti-CD19 antibody (PE-cy7, BD 552854); anti-CD4 antibody (APC-cy7, BD 552051); anti-NK1.1 antibody (BV421, BD 562921); anti-CD45 antibody (BV510, BD 563891); anti-CD11b antibody (PP5.5, BD 550993); anti-CD11c antibody (PE-cy7, BD 558079); anti-F4/80 antibody (APC, BD 566787); and anti-MHCII antibody (APC-cy7, Biolegend 107628).
For flow cytometry, we used the following antibodies: anti-TCRβ antibody (FITC, BD 553170); anti-TCRγδ antibody (PE, BD 553178); anti-CD8a antibody (PP5.5, BD 551162); anti-CD19 antibody (PE-cy7, BD 552854); anti-CD4 antibody (APC-cy7, BD 552051); anti-NK1.1 antibody (BV421, BD 562921); anti-CD45 antibody (BV510, BD 563891); anti-CD11b antibody (PP5.5, BD 550993); anti-CD11c antibody (PE-cy7, BD 558079); anti-F4/80 antibody (APC, BD 566787); and anti-MHCII antibody (APC-cy7, Biolegend 107628).
10
Quantification of Hepatic CD8+ Cells
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Livers were dissected and fixed in 4 % PFA O/N at 4° C. Livers were transferred to 15 % sucrose in PBS and 30 % sucrose in PBS at 4° C until tissue sunk and, then embedded in Tissue-tek OCT. For immunofluorescence, 5 μm OCT tissue cryosections were stained with anti-Cd8 antibody (#550281, BD), incubated with donkey anti-rat Alexa Fluor 595 secondary antibody (#A21209, Thermofisher) and counterstained with DAPI. Random field (6 per mice) were collected using a Nikon Eclipse 90i microscope and positively stained cells were quantified using ImageJ Software (NIH).
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