Excised tumors were minced into pieces, and then dissociated by passing through a 70 μm cell strainer to obtain single cell suspension. Spleens were processed by mechanical dissociation, followed by lysis of red blood cells (Cat#A10492-01, Gibco). Ficoll-Paque PLUS (Cat#17-1440-03, GE Healthcare Life Sciences) was added to the bottom of the conical tubes containing single cell suspension in RPMI-1640. Density gradient centrifugation was performed to purify immune cells. Rare sample with inadequate number of TILs is excluded from further processing. Flow cytometry antibodies include: CD3 (17A2, BD Biosciences), CD4 (RM4-5, Biolegend), CD8 (53-6.7, Biolegend), CD366 (RMT3-23, Biolegend), CD279 (29F.1A12, Biolegend), CD16/32 (93, eBioscience), MHC-class II (M5/114.15.2, eBioscience), CD86 (GL1, eBioscience), tetramer recognizing HLA-A*0201-restricted EGFR 854L.ILDFGLAKL (NIH tetramer core), tetramer recognizing H-2Db-restricted HPV16 E7 epitope RAHYNIVTF (NIH tetramer core), and viability dye (Cat#65-0865-14, eBioscience). All data were analyzed using FlowJo.
Mhc class 2 m5 114
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MHC-class II (M5/114.15.2) is a laboratory antibody used to detect major histocompatibility complex (MHC) class II molecules in various biological samples. It recognizes a specific epitope on the MHC class II complex. This antibody can be utilized in techniques such as flow cytometry, immunohistochemistry, and Western blotting to identify and analyze cells expressing MHC class II proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (1 mentions)
Viability dye, by Thermo Fisher Scientific (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
Cd86 gl1, by Thermo Fisher Scientific (1 mentions)
Cd3 17a2, by BD (1 mentions)
Cd4 rm4 5, by BioLegend (1 mentions)
Cd8 53 6, by BioLegend (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Cd4 gk1, by BD (1 mentions)
Cd8α 53 6, by Thermo Fisher Scientific (1 mentions)
Cd38 90, by Thermo Fisher Scientific (1 mentions)
Il 10 jes5 16e3, by Thermo Fisher Scientific (1 mentions)
Cd11c hl3, by BD (1 mentions)
Il 17a tc11 18h10, by BD (1 mentions)
Cd64 x54 5 7, by BioLegend (1 mentions)
Cd103 2e7, by BioLegend (1 mentions)
Cd95 jo2, by BD (1 mentions)
4 protocols using mhc class 2 m5 114
1
Tumor-Infiltrating Lymphocyte Isolation
2
Multiparametric Flow Cytometry of Immune Cells
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Monoclonal antibodies used were CD11c (HL3; BD), CD103 (2E7; BioLegend), CD11b (M1/70; eBioscience), CD64 (X54‐5/7.1; BioLegend), MHC class II (M5/114.15.2; eBioscience), CD45 (30‐F11; BioLegend), CD45R (RA3‐6B2; BD), CD8α (53‐6.7; eBioscience), CD197 (4B12; eBioscience), Plet1 (1D4), CD38 (90; eBioscience), CD19 (1D3; eBioscience), CD95 (Jo2; BD), Donkey anti‐rat IgG (Life Technologies), CD4 (GK1.5; BD), Foxp3 (Fjk‐16s; eBioscience), IL‐10 (JES5‐16E3; eBioscience), IFNγ (XMG1.2; BD), and IL‐17A (TC11‐18H10; BD Pharmingen). Zombie Aqua Fixable viability kit (BioLegend) was used for dead cell exclusion.
3
Multicolor Flow Cytometry Analysis
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Fluorescein isothiocyanate (FITC) or phycoerythrin-conjugated mAbs directed to CD11c (HL3), CD40 (HM40-3), CD80 (16-10A1), CD86 (GL1), MHC class I (AF6-88.5.5.3) and MHC class II (M5 / 114.15.2) were from eBioscience (San Diego, CA, USA). In all cases, isotype-matched control antibodies were used, and a gate (R1) was defined in the analysis to exclude all nonviable cells and debris, based on size and propidium iodide staining. The analysis was performed using a PartecCyflow Space (Sysmex, UK) flow cytometer, and the FlowJo software (Treestar). The results are expressed as the mean fluorescence intensity or as the percentage of positive cells. Data were statistically compared using the Kruskal-Wallis test with Dunn's multiple comparisons post-test; p values of < 0.05 were considered to be significant.
4
Multiparameter Cell Sorting and Analysis
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Multiparameter assessment and cell sorting were performed using LSR Fortessa, BD FACS ARIA II and ARIA III (BD Biosciences) and data were analyzed with FlowJo software (TreeStar). After blocking the FcgIII/II receptors by incubation with homemade anti-CD16/32 (2.4G2), single-cell suspensions were incubated with the indicated fluorochromeconjugated or biotinylated monoclonal antibodies in FACS buffer (PBS containing 2% FCS and 2 mM EDTA) and then washed twice before detection. Monoclonal antibodies specific to mouse CD45 (30-F11), CD11c (N418), F4/80 (BM8), CD11b (M1/70), Siglec-F (E50-2440, BD Biosciences), CD45.1 (A20), CD45.2 (104), Ly6C (HK1.4), GM-CSFRa (698423, R&D), CD64 (X54-5/7.1) and MHC class II (M5/114.15.2, eBioscience) were purchased from BioLegend unless otherwise stated. Dead cells were excluded using the live/dead marker eFluor780 (eBioscience).
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