On day 7, the tumour-bearing mice were treated with 3 mg/kg SZU-101 or 4 mg/kg DOX by intraperitoneal administration. Mouse spleens were collected at 2, 4 or 24 h post treatment on day 8, and the splenocytes were prepared by removing the red blood cells with RBC lysis buffer (BioLegend) after separating the cells through a 70-μm cell strainer. Approximately 1 × 106 cells were stained with corresponding florescence antibodies and analysed by FACSCalibur flow cytometry (BD Biosciences). The antibodies for flow cytometry were purchased from BioLegend and included the following: anti-CD11c-Alexa488, anti-CD86-PerCP/Cy5.5, anti-CD80-Alexa647, anti-CD3-Alexa488, anti-CD19-Alexa647 and anti-CD69-PerCP/Cy5.5.
Anti cd86 percp cy5
Manufactured by BioLegend
Sourced in United States
Anti-CD86-PerCP/Cy5.5 is a fluorochrome-conjugated antibody that binds to the CD86 surface antigen. CD86 is a co-stimulatory molecule expressed on antigen-presenting cells and plays a role in T cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometry, by BD (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Anti cd3 alexa488, by BioLegend (1 mentions)
Anti cd69 percp cy5, by BioLegend (1 mentions)
Anti cd11c alexa488, by BioLegend (1 mentions)
Fc block, by BD (1 mentions)
Anti cd19 pe, by BioLegend (1 mentions)
Pe cy7 anti cd40, by BioLegend (1 mentions)
Anti cd19 fitc, by BioLegend (1 mentions)
Anti cd38 fitc, by BioLegend (1 mentions)
Flow cytometry staining buffer, by BD (1 mentions)
Anti cd69 fitc, by BioLegend (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Human pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 protocols using anti cd86 percp cy5
1
Immunomodulatory Effects of SZU-101 and Doxorubicin
2
Multicolor Flow Cytometry Analysis of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed in flow cytometry staining buffer (BD Pharmingen), treated with Fc block (BD Bioscience) and stained for flow cytometric analyses of extracellular markers using fluorochrome‐conjugated antibodies according to the manufacturer's instructions. Antibodies included anti‐CD19‐PE or anti‐CD19‐FITC, anti‐CD5‐BV421 and the activation markers anti‐HLA‐DP, DQ, DR Alexa Fluor 647 or anti‐HLA‐DP/DQ/DR‐FITC, anti‐CD86‐PerCP‐CY5.5, anti‐CD69‐FITC and anti‐CD40‐PE‐Cy7 (BioLegend); markers of maturation anti‐CD38‐FITC or anti‐CD38‐APC and CD24‐APC‐Cy7 (BioLegend); or CD22‐FITC and sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐10‐APC (BioLegend). Immunoglobulin isotype‐matched control antibodies were used to confirm specificity of staining. Extracellular staining was conducted for 30 min in the dark at 4°C, before the cells were washed and analysed by flow cytometry. To correct for inter‐assay variation, ratios were calculated of staining intensity in treated versus control cultures.
3
Differentiation of Dendritic Cells from PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Complete media (CM) including RPMI 1640 (Gibco, USA, NY) that contains 10% Fetal Bovine Serum (FBS) (Gibco, USA, NY), Streptomycin 100 μg/mL, Penicillin 100 IU/mL (Gibco, USA, NY), 2 mmol/L of L-glutamine (Gibco, USA, NY). 2-mercaptoethanol (2ME) was ordered from Gibco (USA, NY). Recombinant human granulocyte macrophage colony stimulating factor (rh GM-CSF) was purchased from Sigma Chemical Co (Munich, Germany) and recombinant human interleukin-4 (rh IL-4) from eBioscience (CA, USA). Lipopolysaccharide (LPS) was ordered from Sigma Chemical Co (Munich, Germany). Carboxyfluorescein succinimidyl ester (CFSE) cell labeling kit was obtained from BioLegend (San Diego, United States). Human pan T cell isolation Kit was purchased from MiltenyiBiotec, Germany. Antibodies used to phenotype the cells were anti-HLA-DR-APC and anti-CD86- PerCP-cy5.5 from BioLegend (San Diego, United States), anti-CD40-CF-blue, anti-CD11c-FITC, and anti-CD14-FITC from Immunostep (Salamanca, Spain). Ficoll was obtained from Sigma Chemical Co (Munich, Germany). Bradford protein assay kit was purchased from Bio-Rad, (Hercules, CA).
4
Multiparametric Flow Cytometry Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the staining of cell surface molecules, cells were resuspended in FACS buffer (PBS, 1% BSA, 0.025% NaN3) and Fc receptors were blocked with 1/200 diluted anti-mouse CD16/32 antibodies (Fc block) (Tonbo Biosciences, San Diego, CA, USA) for 10 min on ice.
BMDCs and splenocyte single cell suspensions were stained with the following 1/400 diluted fluorochrome conjugated anti-mouse Abs: anti-CD11c-APC, anti-MHC (I-A/I-E)-PE-Cy7, anti-MHC (I-A/I-E)-APC-Fire750, anti-CD80-Pacific Blue, anti-CD86-PerCp Cy5.5, anti-CD86-PE-Cy7, anti-F4/80-PE, anti-CD11b-PerCp-Cy5.5 (Biolegend, San Diego, CA, USA) and incubated in the dark on ice for 40 minutes. The cells were fixed with Cytofix-fixation buffer (BD Biosciences) for 15 min on ice and cell fluorescence was assed using Fortessa (BD Biosciences). The data were analyzed with FlowJo software version 10. Gating strategy for BMDCs: FSC-A: SSC-A > singlets > CD11c + MHC II + > MHC II + CD86 + , MHC II + CD80 + .
Gating strategy for DCs from splenocytes: FSC-A: SSC-A > singlets > F4/80 -> CD11b + CD11c + > MHC II + CD86 + > MHC II + CD80 + . Gating strategy for lymphocytes: FSC-A: SSC-A > singlets > live-dead > CD4 + CD8 -; CD4 -CD8 + > CD44 + .
BMDCs and splenocyte single cell suspensions were stained with the following 1/400 diluted fluorochrome conjugated anti-mouse Abs: anti-CD11c-APC, anti-MHC (I-A/I-E)-PE-Cy7, anti-MHC (I-A/I-E)-APC-Fire750, anti-CD80-Pacific Blue, anti-CD86-PerCp Cy5.5, anti-CD86-PE-Cy7, anti-F4/80-PE, anti-CD11b-PerCp-Cy5.5 (Biolegend, San Diego, CA, USA) and incubated in the dark on ice for 40 minutes. The cells were fixed with Cytofix-fixation buffer (BD Biosciences) for 15 min on ice and cell fluorescence was assed using Fortessa (BD Biosciences). The data were analyzed with FlowJo software version 10. Gating strategy for BMDCs: FSC-A: SSC-A > singlets > CD11c + MHC II + > MHC II + CD86 + , MHC II + CD80 + .
Gating strategy for DCs from splenocytes: FSC-A: SSC-A > singlets > F4/80 -> CD11b + CD11c + > MHC II + CD86 + > MHC II + CD80 + . Gating strategy for lymphocytes: FSC-A: SSC-A > singlets > live-dead > CD4 + CD8 -; CD4 -CD8 + > CD44 + .
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!