B16-F10 tumor material was collected for FACS analysis and single-cell suspension obtained with digestion mixture [0.5 mg/mL collagenase A, (Sigma Aldrich, MO, USA); 0.2 mg/mL hyaluronidase type V, (Sigma Aldrich, MO, USA); 0.02 mg/mL DNase I, (Roche Diagnostic GmbH, Germany); per each 0.25 g of tumor tissue). Red blood cells were lysed using 0.15 M ammonium chloride solution (Sigma Aldrich, MO, USA)]. Dead cells were removed by centrifugation on Lympholyte-M gradients (Cedarlane, Canada). Level of T lymphocytes was determined in homogenous single-cell suspension. To identify the subpopulations of T lymphocytes, the following antibodies were used: PE-Cy™7-CD3e, PE-CD4 and FITC-CD8a (BD, Franklin Lakes, NJ, USA). NK cells were identified with an anti-mouse CD49b antibody (Biosciences, CA, USA). Gate parameters dividing negative from positive cells were chosen based on isotype antibody control probes (Jarosz et al. 2013 (link)).
Cd8a fitc
Manufactured by BD
Sourced in United States
CD8a-FITC is a fluorochrome-conjugated antibody that binds to the CD8a surface protein, which is commonly expressed on cytotoxic T cells. It is used for the identification and enumeration of CD8+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe, by BD (4 mentions)
Cd8a pe, by BD (4 mentions)
Cd4 apc, by BD (4 mentions)
Cd4 fitc, by BD (3 mentions)
Cd45 apc cy7, by BD (2 mentions)
Cd3 apc cy7, by BD (2 mentions)
Cd3 apc, by BD (2 mentions)
Cd45 apc, by BD (2 mentions)
Cd11c fitc, by BD (2 mentions)
Cd45 pe, by BD (2 mentions)
Cd86 pe, by BD (2 mentions)
Gr1 apc, by BD (2 mentions)
Cd25 pe, by BD (2 mentions)
Ifn γ pe, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Ammonium chloride solution, by Merck Group (1 mentions)
Hyaluronidase type 5, by Merck Group (1 mentions)
Lympholyte m gradient, by Cedarlane (1 mentions)
Pe cy 7 cd3e, by BD (1 mentions)
16 protocols using cd8a fitc
1
Isolation and Identification of Tumor Immune Cells
2
Multicolor Flow Cytometry of Immune Cells
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Cells were washed in FACS buffer (0.05% BSA, 2 mM EDTA in PBS, pH 7.4) blocked using anti-CD16/32 (1 µg; Biolegend) for 15 mins at 4°C, followed by 30 min antibody staining for the surface markers with isotype controls. Cells were again washed and resuspended in 200 µL of FACS buffer without fixation. Antibodies used are listed in Table 1
Intracellular staining was performed as per manufacturer’s instructions using eBioscience FOXP3/Transcription Factor Staining Buffer Set (invitrogen). Briefly, after the last wash of the extracellular staining protocol, cells were fixed in Fixation/Permeabilization Buffer for 30 min at room temperature. Cells were washed and resuspended in Permeabilization Buffer; intracellular antibodies were added for 30 min in room temperature. Cells were again washed in Permeabilization Buffer and resuspended in FACS buffer for flow cytometric analysis.
Data were acquired using BD Fortessa X20 cytometer and Diva software (BD Biosciences) and then analysed using FlowJo (Tree Star Inc, USA) software. Fluorescence minus One (FMO) controls were used for gating and single stains for compensation were obtained using UltraComp eBeads™ Compensation Beads (InVitrogen) for liver, spleen and blood panels and single stained cell samples for peritoneal cells.
Intracellular staining was performed as per manufacturer’s instructions using eBioscience FOXP3/Transcription Factor Staining Buffer Set (invitrogen). Briefly, after the last wash of the extracellular staining protocol, cells were fixed in Fixation/Permeabilization Buffer for 30 min at room temperature. Cells were washed and resuspended in Permeabilization Buffer; intracellular antibodies were added for 30 min in room temperature. Cells were again washed in Permeabilization Buffer and resuspended in FACS buffer for flow cytometric analysis.
Data were acquired using BD Fortessa X20 cytometer and Diva software (BD Biosciences) and then analysed using FlowJo (Tree Star Inc, USA) software. Fluorescence minus One (FMO) controls were used for gating and single stains for compensation were obtained using UltraComp eBeads™ Compensation Beads (InVitrogen) for liver, spleen and blood panels and single stained cell samples for peritoneal cells.
3
Multicolor Flow Cytometry for Immune Cell Analysis
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Blood was collected with heart puncture and transferred into a Na EDTA-containing tube. Single cell suspensions were prepared from spleens and BM as described (Heo, 2005 ). Whole blood (50 μl) was stained and erythrocytes were lysed with FACSlyse (BD Biosciences, San Jose) before analysis. Splenocytes and BM cells were counted, and 1 × 106 cells were used for staining followed by FACSlyse and analysis by six-color flow cytometry with a FACSCanto flow cytometer (BD Biosciences), and the following fluorochrome conjugated Abs: PerCP Cy5.5-CD45, APC Cy7-CD45, PerCP Cy5.5-CD4, PE-CD4, FITC-CD4, FITC-CD8a, PE-CD8a, PerCP-CD8a, FITC-CD3, APC-CD3e, PerCP-CD3e, PE Cy7-CD24, FITC-CD43, PerCP-CD249- APC Cy7-B220, PE -IgD, APC-IgM, APC Cy7-CD3, PE Cy7-CD19, PerCP cy5.5-CD93, APC-CD138, PE-CXCR4, FITC-MHC-II, PE-CD19, FITC-CD19, PE Cy7-CD19, APC-CXCR5, PE Cy7-PD1, PerCP Cy5.5-CD138 and anti-CD16/32 (Fc block). The Abs were purchased from BD Pharmingen (San Diego, CA), Biolegend (San Diego, CA), or eBiosciences (Thermo Fisher Scientific, MA, USA). Frequencies and numbers of populations in the blood and spleens of B6 and BTBR mice were gated based on FSC-A and SSC-A, followed by gating out the doublets and finally gated on CD45+ cell. Data were analyzed by Flow Jo-V10.
4
Tumor-Infiltrating Lymphocyte Isolation
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Spleen cell suspension was prepared as mentioned above for cytokine analysis. TILs were isolated from freshly dissected tumors. Briefly, tumors were washed with PBS, minced using a razor blade and incubated with collagenase IV (Sigma Aldrich, St Louis, MI, USA, 0.5 mg/mL in RPMI) for 1 h at 37 ·C. Digested tissues were then filtered through a 100 μm pore size strainer to obtain a single-cell suspension and cells were washed once before staining. Approximately 106 cells were suspended in PBS and incubated with CD16/CD32 Fc block antibody (Cat553141, BD Biosciences, San Jose, CA, USA) for 20 min at 4 ·C. Cells were washed with PBS and incubated with 1 μL of the appropriate fluorochrome-conjugated antibody for 40 min at 4 ·C. Cells were washed with PBS and incubation with conjugated antibodies was performed. Antibodies used in these analyses were from BD Biosciences (San Jose, CA, USA): FITC-CD45 (Cat 553772), FITC-CD8a (Cat 553031), PE-CD8a (Cat 553033), PE-CD4 (Cat 553730), PE-CD3e (Cat 553063), APC-CD4 (Cat 353051) and APC-CD49b (Cat 560628). Finally, cells were washed twice with PBS before flow cytometry analysis (Calibur, BD Biosciences, San Jose, CA, USA).
5
Immunofluorescence and Flow Cytometry Analysis of Tumor-Infiltrating Lymphocytes
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For immunofluorescence studies, tumors were fixed for 24 h in 4% paraformaldehyde (PFA), followed by overnight incubation in a 30% sucrose solution. Tumors were then snap-frozen in optimal cutting temperature compound (OCT) and 5 μm sections were obtained. For CD8 (Cell Signaling, Danvers, MA, USA, 98941) and CD11c (Cell Signaling, 97585S) staining, slides were fixed for 10 min in 4% PFA before antigen retrieval in citrate buffer (pH 5.7) with 0.5% Triton. After the addition of an AlexaFluor-488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA, A11034), slides were scanned (3DHistech Pannoramic P250 Flash III, Budapest, Hungary, 20X) and the extent of CD8 and CD11c staining was quantified over the total tumor area using Visiopharm software. For TIL analysis by flow cytometry, tumors were mechanically dissociated and single cell suspension was obtained by passing them through a 70 µm strainer. Cells were stained with CD45-BV510 (BD Pharm, 513151), CD3-APC (BD Pharm, 553066), CD8a-FITC (BD Pharm, 553031) and CD4-BV421 (BD Pharm, 553066) for 15 min RT.
6
Comprehensive Immune Cell Profiling
7
Tumor Dissociation and Immune Cell Profiling
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After cervical dislocation of mice, tumors were excised, minced and then incubated in an enzyme mixture (7.5 mg collagenase type I, 2.5 mg hyaluronidase type I-S (both Sigma-Aldrich, Germany) in 10 ml PBS) at 37°C for 1 h. After filtration of the cell suspension through a 100 μm nylon mesh erythrocytes were lyzed in 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2-EDTA (pH 7.4, 4°C, 8 min) and washed with PBS.
For FACS analysis cells were stained using the following anti-mouse antibodies: anti-CD4-FITC, CD8a-FITC, CD11b-FITC, CD11c-FITC, CD45-APC (all BD, Germany), CD25-PE (Miltenyi Biotec, Germany), as well as appropriate isotype controls. Cells were fixed in 4% (v/v) paraformaldehyde after staining. Analysis was performed on a BD FACSCanto II using the FACS Diva Software (both BD, Germany). Gates of immune cell subpopulations were defined by staining with respective isotype control antibodies and fractions are given as percentage of CD45+ immune cells.
For the IFN-γ-ELISpot 2 × 105 cells of the tumor were used and protocol was run according to the manufacturer’s specification (R&D Systems, Germany). The number of IFN-γ secreting cells was determined by counting the number of visible spots on the plate.
For FACS analysis cells were stained using the following anti-mouse antibodies: anti-CD4-FITC, CD8a-FITC, CD11b-FITC, CD11c-FITC, CD45-APC (all BD, Germany), CD25-PE (Miltenyi Biotec, Germany), as well as appropriate isotype controls. Cells were fixed in 4% (v/v) paraformaldehyde after staining. Analysis was performed on a BD FACSCanto II using the FACS Diva Software (both BD, Germany). Gates of immune cell subpopulations were defined by staining with respective isotype control antibodies and fractions are given as percentage of CD45+ immune cells.
For the IFN-γ-ELISpot 2 × 105 cells of the tumor were used and protocol was run according to the manufacturer’s specification (R&D Systems, Germany). The number of IFN-γ secreting cells was determined by counting the number of visible spots on the plate.
8
Flow Cytometry Analysis of Immune Cells
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Immune cells were analyzed by flow cytometry, as previously described. To prevent nonspecific binding to the Fcγ receptor, single-cell suspensions were first incubated with an anti-CD16/32 antibody. Fixable viability stain 510 (564406, BD Biosciences) and the following antibodies were used for flow cytometry: CD45-APC-Cy7 (557659, BD Biosciences), CD45-PE (553081, BD Biosciences), CD11b–fluorescein isothiocyanate (FITC; 557396, BD Biosciences), CD11b-PE-Cy7 (552850, BD Biosciences), CD11b-PE (553311, BD Biosciences), CD64-APC (558539, BD Biosciences), Ly6C-PE-Cy7 (560593, BD Biosciences), Ly6C-APC (Invitrogen, 17-5932-82), Ly6G-APC (560599, BD Biosciences), Ly6G-FITC (551460, BD Biosciences), F4/80-BV421 (565411, BD Biosciences), CD3e-BV421(564008, BD Biosciences), CD3e-PE (12-0031-81, eBioscience), CD4-APC(553051, BD Biosciences), CD8a-PE (553033, BD Biosciences), CD8a-FITC (553031, BD Biosciences), MHCII-BV421 (743870, BD Biosciences), CCR2-PE (FAB5538P, R&D Systems), CD86-PE (553692, BD Biosciences), CD206-APC (565250, BD Biosciences), and CD31-PE (553373, BD Biosciences). Flow cytometry and cell sorting were performed using a FACSAria flow cytometer (BD Biosciences). Data are expressed as the absolute number of cells per milligram of tissue and were analyzed using the FlowJo software.
9
Phenotypic Analysis of Rodent Tumor Cells
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Rodent tumors were harvested and resuspended as single cells in phosphate-buffered saline (PBS)/0.3% bovine serum albumin. Cells were washed and incubated with the following antibodies: CD45-APC-Cy7 (BioLegend, clone 30-F11, catalog no. 103116, lot no. B242535), CD3e-PE (BD, clone 145-2C11, catalog no. 553061, lot no. 22126), GR1-PerCP (BD, clone Ly-6G/Ly-6C, catalog no. 552093, lot no. 73108), TER119-APC (BD, clone Ter-119, catalog no. 557909, lot no. 42622), B220–Alexa Fluor 647 (BD, clone RA3-6B2, catalog no. 557683, lot no. 22218), CD4-PerCP (BD, clone L3T3, catalog no. 553654, lot no. 60912), and CD8a-FITC (BD, clone Ly-2, catalog no. 553030, lot no. 46675), as indicated. Cells were analyzed on a custom-built LSR II flow cytometer (BD). Data compensation and analysis were performed using noncommercial software developed in our laboratory (73 (link)).
10
Flow Cytometry Analysis of Tumor Infiltrates
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