MSC previously isolated from ICR mouse bone marrow were obtained from the culture collection of the Immunology Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. Frozen vials stored in liquid nitrogen were thawed at 37°C and cultured in MSC complete medium (high glucose DMEM (GIBCO Invitrogen, CA, USA), supplemented with 15% (v/v) MesenCult® Mesenchymal Stem Cell Stimulatory Supplements (Mouse) (Stemcell™ Technologies, Canada), 1% penicillin and streptomycin (iDNA), 250 μg/ml fungizone (GIBCO Invitrogen, CA, USA), 2.0 mM GlutaMaX and 1.5 g sodium bicarbonate) at 37°C, 5% CO2 in a humidified incubator. Cells were routinely sub-cultured using 0.25% Trypsin-EDTA (GIBCO Invitrogen, CA, USA) before reaching 90% confluency and immunophenotyped using a panel of markers comprising CD106, CD45, CD44, CD11b, Sca-1, MHC-I and MHC-II (all from Becton Dickinson, BD, San Jose, CA, USA) [1 (link)]. MSC phenotype was confirmed by positivity to CD106, CD44, Sca-1 and MHC-I and negativity to CD45, CD11b and MHC-II.
Mhc 1
Manufactured by BD
Sourced in United States
The MHC-I is a lab equipment product that serves as a core component in the analysis of major histocompatibility complex class I (MHC-I) molecules. MHC-I plays a crucial role in the immune system by presenting peptide fragments to cytotoxic T cells. The MHC-I product provides the necessary tools and technologies for researchers to study this fundamental immunological process.
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Lab products found in correlation
Mhcii, by BD (4 mentions)
Cd11b, by BD (2 mentions)
Canto 2, by BD (2 mentions)
Cd62l, by BD (2 mentions)
F4 80, by BD (2 mentions)
Sca 1, by BD (1 mentions)
Cd106, by BD (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Mhc 2, by BD (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Mz10f, by Leica (1 mentions)
Mhc 1, by Thermo Fisher Scientific (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
Anti active caspase 3, by BD (1 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (1 mentions)
Axiocam 503 color microscope, by Zeiss (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
12 protocols using mhc 1
1
Isolation and Characterization of Mouse Mesenchymal Stem Cells
2
Quantification of Disseminated PDX Cells
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Following 8–14 weeks post orthotopic transplants, or ~8 weeks post i.c. or i.v. injections, primary tumors and peripheral tissues including lungs, lymph nodes, bone marrow, peripheral blood, and brains were harvested, dissociated to single cells and stained with fluorescently conjugated antibodies for CD298 (Biolegend, Cat. no. 341704) and MHC-I (eBioscience, Cat. no. 17-5957-82), and flow cytometry was used to analyze and quantify disseminated CD298+MHC-I− PDX cells using the BD FACSAria Fusion cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) as described previously26 (link). Primary tumor volumes were calculated using caliper measurements with the equation: volume of an ellipsoid = 1/2(length × width2). Whole-mount images of primary tumors and lungs were taken with a Leica MZ10 F modular stereomicroscope (Leica Microsystems, Buffalo Grove, IL, USA).
3
Multiparameter Flow Cytometry of Immune Cells
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Spleen and lymph nodes were isolated, and single-cell suspensions were obtained by crushing the tissues through 70 µm filters. The spleen and blood samples were subjected to red blood cell lysis (red blood cell lysis buffer: 8.4 g of NH4Cl and 0.84 g of NaHCO3 per liter of distilled water). Single cell suspensions were stained with fluorescently labeled antibodies (CD45, CD3, CD4, CD8, F4/80, Ly6C, Ly6G, MHCI, MHCII, CD44, CD62L, CD19, CD11C, NK1.1, all from BD Biosciences, San Diego, CA, USA). Nonspecific binding was prevented by the pre-incubation of the cells with a Fc receptor-blocking antibody CD16/32 (Ebioscience, Vienna, Austria). Flow cytometry was performed on a BD Canto II (BD Biosciences), and analysis was performed using the Flowjo software version 10 (Tree star, Ashland, OR, USA).
4
Immunostaining Analysis of Frozen Tissue Samples
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Histological analysis was performed on snap frozen tissue. Tissue sections were fixed in acetone or 10% neutral buffered formalin, blocked with 5% FCS/0.3% Triton-X in PBS and stained with anti-active Caspase 3 (BD Biosciences), cleaved Caspase 8, cleaved Caspase-9, p-CREB (Ser 133), p-AMPK (Thr172) (all from Cell Signaling), CD8 and MHC-I (both from BD Biosciences) followed by incubation with the appropriate secondary antibodies. Cy3-conjugated anti-rabbit secondary antibodies were used for immunofluorescence. HRP-linked anti-rabbit secondary antibodies were used for conventional staining, which were visualised with the Peroxidase Substrate (ImmPACT NovaRED). Images were taken with an Axio Observer Z1 fluorescence microscope or Axiocam 503 color microscope (ZEISS) and quantified using Image J using the fluorescence intensity (MFI) per area as previously described [72 (link)].
5
Tumor-Infiltrating Lymphocyte Immunohistochemistry
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Tumor masses were blocked after the sacrifice of tumor-bearing mice.
Tumor-infiltrating lymphocytes were indicated by hematoxylin & eosin
(H&E) staining. For immunohistochemical staining, all of the
paraffin-embedded sections were cut to 5-µm thickness, deparaffinized with
xylene, and dehydrated with graded ethanol. Antigen recovery was performed in
heat-activated antigen retrieval pH 9 (Dako, Carpinteria, CA, USA), after which
the specimens were incubated with 3% H2O2 for 15 min.
Non-specific binding was blocked with protein block (Dako) for 20 min at room
temperature. The sections were incubated with FasL (PharMingen), MHC I
(PharMingen), CD301 (PharMingen), and T/Tn (Chicago Medical School) at 1:50
dilution for 2 h. Subsequently, the sections were incubated with
EnVision+ Dual Link System-HRP (Dako) for 30 min, visualized with
3,3-diaminobenzadine for 10 min, and washed and counterstained with hematoxylin.
Appropriate negative controls were concurrently performed. All of the slides
were reviewed by a pathologist.
Tumor-infiltrating lymphocytes were indicated by hematoxylin & eosin
(H&E) staining. For immunohistochemical staining, all of the
paraffin-embedded sections were cut to 5-µm thickness, deparaffinized with
xylene, and dehydrated with graded ethanol. Antigen recovery was performed in
heat-activated antigen retrieval pH 9 (Dako, Carpinteria, CA, USA), after which
the specimens were incubated with 3% H2O2 for 15 min.
Non-specific binding was blocked with protein block (Dako) for 20 min at room
temperature. The sections were incubated with FasL (PharMingen), MHC I
(PharMingen), CD301 (PharMingen), and T/Tn (Chicago Medical School) at 1:50
dilution for 2 h. Subsequently, the sections were incubated with
EnVision+ Dual Link System-HRP (Dako) for 30 min, visualized with
3,3-diaminobenzadine for 10 min, and washed and counterstained with hematoxylin.
Appropriate negative controls were concurrently performed. All of the slides
were reviewed by a pathologist.
6
Comprehensive Immune Cell Profiling
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Spleen and lymph nodes were homogenized, and blood and spleen samples were subjected to red blood cell lysis. The cells were stained with fluorescently labeled surface antibodies (CD45, CD3, CD4, CD8, F4/80, Ly6C, Ly6G, MHCI, MHCII, CD44, CD62L, CD19, CD11C, NK1.1; all from BD Biosciences). For intracellular staining, cells were fixed and permeabilized with fixation/permeabilization buffer (eBioscience) and stained with fluorescent antibodies against Foxp3 (BioLegend). Flow cytometric analysis was performed on a BD Canto II (BD Biosciences).
7
Flow Cytometric Analysis of Cell Surface and Functional Markers
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For surface staining, cells were stained with antibodies (CD3, CD4, CD8, MHC-I, MHC-II from BD Biosciences or Biolegend, dilution 1:200) for 30–45 min in cell isolation buffer in dark tubes before analysis. For NCC function study using CoroNa Green (cell permeable, Molecular Probes), mDCTs were treated with ouabain, bumetanide and amiloride for 30 min at room temperature (RT) followed by loading CoroNa Green at a concentration of 10 μM for 1 h at RT. Cells were washed and re-suspended in PBS containing the same blockers mentioned above for 45 min and analysed in a BD Accuri C6 flow cytometer immediately. Flow cytometry data were analysed using FlowJo software.
8
Immunophenotyping of Stem Cells
9
Multiparametric Flow Cytometry Analysis
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Cells from tumor, spleen, lymph nodes and lung were mechanically dissociated, and the red blood cells were removed by ACK lysis buffer (Lonza). Cells were first blocked with Fc antibody and then labeled with different combinations of antibodies. Data were acquired with an LSR Fortessa flow cytometer (BD Biosciences), and analysis was performed using Flowjo software. Cell sorting was performed using a BD FACSAria cell sorter (BD Biosciences). Fluorochrome conjugated CD4, CD8, CD11b, NK1.1, PD1, NKG2D, CD86, MHC I, MHC II, KLRG1, IFNγ and TNFα were bought from BD Biosciences. PE conjugated OT-1 tetramer were bought from Beckman Coulter, Inc.
10
Characterization and Transplantation of Mesenchymal Stem Cells
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Bone marrow aspiration procedures were performed in accordance with the Helsinki Declaration and were approved by the ethics committee of LUMC. All MSC donors provided written informed consent. Bone marrow-derived MSCs were obtained from three non-cardiac patients undergoing orthopedic surgery. IFN stimulation of MSCs was performed by adding 500U/ml IFN (Sigma-Aldrich Chemie BV, Zwijndrecht, the Netherlands) to the culture medium for 7 days.
Immunophenotyping of cultured MSC was performed using the following primary antibodies: CD90, CD73, MHC-I, CD34, CD45, CD31, CD80, HLA-DR (BD Biosciences, San Diego, USA), and CD105 (Ancell Corp., Bayport, MN, USA). MSCs from passages 4 to 5 were used for transplantation experiments after lentiviral transduction with a human vector expressing the enhanced green fluorescent protein (eGFP) gene. The cells transduced with lentivirus for eGFP, transmitted the eGFP signal in the FITC channel of the FACSCanto II (BD Biosciences, San Diego, CA, USA).
Immunophenotyping of cultured MSC was performed using the following primary antibodies: CD90, CD73, MHC-I, CD34, CD45, CD31, CD80, HLA-DR (BD Biosciences, San Diego, USA), and CD105 (Ancell Corp., Bayport, MN, USA). MSCs from passages 4 to 5 were used for transplantation experiments after lentiviral transduction with a human vector expressing the enhanced green fluorescent protein (eGFP) gene. The cells transduced with lentivirus for eGFP, transmitted the eGFP signal in the FITC channel of the FACSCanto II (BD Biosciences, San Diego, CA, USA).
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