Cells from the joint draining LNs of 6 week old KRN.g7 and IDO2 ko KRN.g7 mice were harvested and cultured for 4 hours with 50 ng/ml PMA, 500 ng/ml ionomycin, and 3 μg/ml brefeldin A. After 4 hours, cells were harvested, surface stained for CD4 and CD8 (eBioscience), fixed and permeabilized (IC Fixation and Permeabilization Buffer, eBioscience), then stained for intracellular IL-21 or isotype control. The samples were acquired on a BDFACSCanto II flow cytometer using FACSDiva software and analyzed with FlowJo software.
Cd4 and cd8
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD4 and CD8 lab equipment products are designed for the detection and analysis of CD4+ and CD8+ T cells, which are important subsets of lymphocytes involved in the immune response. These products provide reliable and accurate measurements to support various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ic fixation and permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Pd l1, by Abcam (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Cd45r b220, by BD (1 mentions)
Foxp3 fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cytofix, by BD (1 mentions)
Cd11b, by BD (1 mentions)
T bet, by Thermo Fisher Scientific (1 mentions)
Cd62l, by BD (1 mentions)
Cd172a, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BD (1 mentions)
Gp100, by Agilent Technologies (1 mentions)
Imager z1 microscope, by Zeiss (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Interleukin 6 (il 6), by PerkinElmer (1 mentions)
Opal 4 color ihc kit, by PerkinElmer (1 mentions)
Granzyme b, by Agilent Technologies (1 mentions)
6 protocols using cd4 and cd8
1
Intracellular IL-21 in KRN.g7 Mice
2
Intracellular IL-21 Expression Analysis
3
Multiparametric Flow Cytometry Analysis
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Cell suspensions were incubated with 24G2 hybridoma supernatant (to block Fc receptors) and stained using immunofluorescence-labeled antibodies against the following antigens: B220/CD45R, CD3, CD19, CD11b, Ly6G, CD21, CD5, CD44, CD86, CD38, CD138, CD24, CD172a (SIRPα), CD69, CD43, CD40, CD45.2, CD93, IgM, TLR7 and CD62L from BD Biosciences, IA/IE (MHC class II), GL7, CD23, Tbet, CD4 and CD8 from eBioscience and CD11c and SiglecH from Biolegend. For intracellular TLR7 staining, cell suspensions were stained for the indicated extracellular antigens, before fixation with Cytofix (BD Biosciences). Cells were then permeabilized with saponin 0.1% and stained for TLR7. Intracellular T-bet staining was performed using the FoxP3 Fixation/Permeabilization buffer (eBioscience).
Flow cytometry was conducted using an LSR2 (BD Biosciences) and data were analyzed with FlowJo (Tree Star). The full gating strategy that used for flow cytometry analysis is presented in Supporting Information Fig. 6. For splenocyte proliferation experiments, erythrocyte-depleted splenocytes were labeled with CellTrace Violet (Life technologies) according to manufacturer's recommendations.
After 4 days of stimulation, cells were harvested and stained with antibodies for analysis by flow cytometry.
Flow cytometry was conducted using an LSR2 (BD Biosciences) and data were analyzed with FlowJo (Tree Star). The full gating strategy that used for flow cytometry analysis is presented in Supporting Information Fig. 6. For splenocyte proliferation experiments, erythrocyte-depleted splenocytes were labeled with CellTrace Violet (Life technologies) according to manufacturer's recommendations.
After 4 days of stimulation, cells were harvested and stained with antibodies for analysis by flow cytometry.
4
Flow Cytometric Characterization of Antigen-Specific T Cells
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Staining of the cell surface was performed on blood samples and splenocytes after red blood cell lysis. Cells were stained in staining buffer for 30 minutes with allophycocyanin (APC)labeled H-2 K b -SIINFEKL tetramers or APC-labeled H-2D b -RAHYNIVTF (E7 49-57 ) and fluorescently labeled antibodies specific for mouse CD3 (BD Biosciences), CD4, and CD8 (eBiosciences). 7-Aminoactinomycin D (7AAD; Life Technologies) was used for the exclusion of dead cells.
Overnight intracellular cytokine analysis of PBMCs was performed after incubating the cells with 2 mmol/L of OVA8 and 2 mmol/L of OVA17 or 2 mmol/L of E7 SLP in presence of brefeldin A (7.5 mg/mL; BD Biosciences). The next day the assay was developed as described (19, 20) .
Overnight intracellular cytokine analysis of PBMCs was performed after incubating the cells with 2 mmol/L of OVA8 and 2 mmol/L of OVA17 or 2 mmol/L of E7 SLP in presence of brefeldin A (7.5 mg/mL; BD Biosciences). The next day the assay was developed as described (19, 20) .
5
Immunophenotyping of High-Grade Gliomas
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Formalin-fixed paraffinembedded (FFPE) sections of primary high-grade gliomas were obtained from 16 patients. HLA class I and programmed deathligand 1 (PD-L1) protein expression and tumor antigen expression were investigated using IHC as described previously (20) (link). Monoclonal antibodies (mAbs) against human HLA class I (OriGene Technologies Inc., Rockville, MD, USA), PD-L1 (Abcam, Cambridge, UK), MAGE-A1 (Thermo Scientific, Flemont, CA, USA), MAGE-A3 (Abnova, Taipei, Taiwan, ROC), WT-1 (DakoCytomation, Glostrup, Denmark), HER1 (DakoCytomation) and gp100 (DakoCytomation) were used as the primary antibodies. The staining was evaluated according to the percentage of tumor cells showing positive membranous staining as follows: score 0, less than 1%; score 1, 1 to 5%; score 2, 5 to 50%; and score 3, more than 50%. Regarding tumor-infiltrating lymphocyte (TIL) staining, monoclonal antibodies against CD4 and CD8 (Thermo Fisher Scientific, Waltham, MA, USA), FoxP3 (Abcam, Cambridge, UK, and CD204 (TransGenic Inc., Kobe, Japan) were used.
6
Tumor-Infiltrating Immune Cell Profiling
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For the tumor-infiltrating immune cells, antibodies against CD4 and CD8 (Thermo Fisher Scientific, Waltham, MA, USA), CD204 (Transgenic Inc., Kobe, Japan), IL-6 (Abcam, Cambridge, UK), TGF-beta1 (R and D Systems, Inc., Minneapolis, MN, USA), cytokeratin (AE1 and AE3, Nichirei Bio., Tokyo, Japan), FoxP3 (Abcam), Granzyme B (Dako, Glostrup, Denmark) and PD-L1 (Abcam) were purchased and used for the IHC and immuno-fluorescence analyses. For reference, the staining level of tumor-infiltrating immune cells was assessed by a semiquantitative estimation of the density of CD8+ T cells inside the tumor site as follows: score 0, no or sporadic CD8+ T cells; score 1, moderate number of CD8+ T cells; score 2, abundant CD8+ T cells; and score 3, highly abundant CD8+ T cells [20 (link)].
For immuno-fluorescence staining, the TGF-beta1, IL-6 and CD204 stains were conducted using an Opal 4-color IHC kit (PerkinElmer Inc., Waltham, MA, USA) and evaluated on a fluorescent Zeiss imager Z1 microscope (Carl Zeiss, Oberkochen, Germany).
For immuno-fluorescence staining, the TGF-beta1, IL-6 and CD204 stains were conducted using an Opal 4-color IHC kit (PerkinElmer Inc., Waltham, MA, USA) and evaluated on a fluorescent Zeiss imager Z1 microscope (Carl Zeiss, Oberkochen, Germany).
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