Briefly, to isolate MDSCs from SA penile tumors, tumors were harvest and digested by Mouse Tumor Dissociation kit (Miltenyi Biotec) to obtain the single cells. Total MDSCs were isolated using Mouse Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec). Total spleen T cells were isolated from spleen of 2-month-old male C57BL/6 mice (Jackson Laboratory) using the mouse Pan T Cell Isolation Kit II (Miltenyi Biotec). To assess the suppressive activities of MDSCs on T cell proliferation, we first labeled the T cells with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen). The labeled T cells were stimulated in an antigen-nonspecific manner with anti-CD3 and anti-CD28 antibodies following eBioscience’s protocol. MDSCs were added at different ratios to T cells. CFSE intensity was quantified 72 h later with BD LSRFortessa Cell Analyzer. Viable CD3+ T cells with CFSE peaks located left of the highest peak (i.e. no proliferation, thus no decline of CFSE intensity) were counted as CFSElow (i.e. proliferative) cells.
Mouse myeloid derived suppressor cell isolation kit
Manufactured by Miltenyi Biotec
The Mouse Myeloid-Derived Suppressor Cell Isolation Kit is a laboratory product designed for the isolation of myeloid-derived suppressor cells (MDSCs) from mouse samples. It provides the necessary reagents and protocols for the enrichment and purification of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Super macs separator, by Miltenyi Biotec (2 mentions)
Facsaria cell sorter, by BD (2 mentions)
Mouse tumor dissociation kit, by Miltenyi Biotec (1 mentions)
Anti cd3 and anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Mouse pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Sunrise remote r microplate reader, by Tecan (1 mentions)
Mtt solution, by Merck Group (1 mentions)
Cck 8 reagent, by Dingguo (1 mentions)
Easysep mouse na ve cd8 t cell isolation kit, by STEMCELL (1 mentions)
Trypan blue 0.5, by Harvard Bioscience (1 mentions)
Rbc lysis buffer 10x, by BioLegend (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by GE Healthcare (1 mentions)
Hbss buffer, by GE Healthcare (1 mentions)
Dnase 1, by Roche (1 mentions)
9 protocols using mouse myeloid derived suppressor cell isolation kit
1
Isolating and Assessing MDSCs from Penile Tumors
2
Suppression Assay of MDSC-Mediated T-cell Proliferation
3
Isolation of Myeloid-Derived Suppressor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions of the spleens from tumor-bearing 615 mice were sorted on Super MACS separator (MiltenyiBiotec) using mouse Myeloid-Derived Suppressor Cell Isolation Kit (MiltenyiBiotec) for gMDSCs and mMDSCs according to manufacturer’s protocol or sorted by FACSAria Cell Sorter (BD Biosciences) for CD3+ T cells.
4
Isolation of MDSC-depleted Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ly6C + Ly6G + MDSC were removed from the spleen cell suspensions prior to in vitro stimulation using the mouse Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. The negative fraction constituted the MDSC-depleted spleen cells population. Efficacy of depletion was > 90% as confirmed by flow cytometry.
5
Cytotoxicity Assay of Anti-Cancer Drugs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AB1‐HA or 3LL cells were seeded into 96‐well plates (2 × 103 cells/100 μl/well) the day before treatment. Following treatment with different concentrations of 5‐FU or PEM for 72 h, 50 μl of MTT solution [2 mg/ml; 3‐(4,5‐dimethylthiazol–2‐yl)‐2,5‐diphenyltetrazolium bromide; Sigma] was added to each well and incubated for 3 h at 37°C. The MTT‐containing medium was discarded, and the dark blue crystals were dissolved by adding 100 μl of DMSO (Wako). The absorbance was read using a SUNRISE Remote R microplate reader (Tecan) at 450 nm and reference wavelengths of 630 nm. AB1‐HA tumor‐bearing mice were sacrificed and their spleens were harvested. MDSCs were isolated from splenocytes using a mouse Myeloid‐Derived Suppressor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. After seeding MDSCs (2 × 103 cells/100 μl/well) into a 96‐well plate the day before treatment, cells were treated with different concentrations of 5‐FU for 72 h and then incubated for 3 h with CCK‐8 reagent (DingGuo Bio) at 37°C. We measured the absorbance at 450 nm with a SUNRISE Remote R microplate reader. The half‐maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 5 software.
6
Isolation of G-MDSCs and M-MDSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ly6G+/Ly6Clow/CD11b+ and Ly6G−/Ly6C high/CD11b+ MDSCs (G-MDSCs and M-MDSCs, respectively) were isolated from single-cell suspensions prepared from the spleens of the recipients. The cells were isolated by magnetically activated cell sorting (MACS) using a mouse myeloid-derived suppressor cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the protocol provided by the manufacturer. The purity of the G-MDSC and M-MDSC populations was >90% as determined by a FACSCalibur.
7
Isolating and Transferring Tumor-Associated MDSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ex vivo cell culture and apoptosis analysis, single-cell suspensions of the spleens from tumor-bearing 615 mice were sorted on Super MACS separator (MiltenyiBiotec) using the mouse Myeloid-Derived Suppressor Cell Isolation Kit (MiltenyiBiotec) for gMDSCs and mMDSCsaccording to the manufacturer's protocol.
For MDSCs in vivo adoptive transfer, single-cell suspensions were prepared from bone marrow of tumorbearing 615 mice, and total MDSCs population (CD11b + Gr-1 + ) were sorted via the FACSAria Cell Sorter (BD Biosciences). 5 × 10 6 bone marrow derived MDSCs were injected intravenously into tumor-bearing 615 mice one hour after each MD5-1 treatment (3 injections in total).
For MDSCs in vivo adoptive transfer, single-cell suspensions were prepared from bone marrow of tumorbearing 615 mice, and total MDSCs population (CD11b + Gr-1 + ) were sorted via the FACSAria Cell Sorter (BD Biosciences). 5 × 10 6 bone marrow derived MDSCs were injected intravenously into tumor-bearing 615 mice one hour after each MD5-1 treatment (3 injections in total).
8
Isolation of Immune Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDSCs were purified either from the spleen or the tumor using the Myeloid-Derived Suppressor Cell Isolation mouse Kit (Miltenyi Biotec) according to the manufacturer's protocol. CD4+CD25+ and CD4+CD25− T cells were magnetically enriched from either the spleen or the tumor using CD4+CD25+ Regulatory T Cell Isolation Kit, mouse (Miltenyi Biotec). CD4+ T cells were harvested after the first step of CD4+CD25+ cell's isolation. CD8+ T cells were isolated from the spleen of naive mice with EasySep™ Mouse Naïve CD8+ T Cell Isolation Kit (StemCell) according to manufacturer's instructions.
9
Isolation and Analysis of Murine Myeloid-Derived Suppressor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used are listed in the Suppl. Table 1. FoxP3 staining Buffer Set was purchased from eBioscience. Collagenases III and IV and Trypan Blue 0.5 % were obtained from Biochrom AG. DNAse I was purchased from Roche. Dulbecco’s PBS Solution (10x), HBSS Buffer, and RPMI 1640 were obtained from PAA Laboratories GmbH. RBC Lysis Buffer (10x) was purchased from BioLegend and Hyaluronidase by Linaris GmbH. CD11b MicroBeads as well as Myeloid-Derived Suppressor Cell Isolation mouse kit were purchased by Miltenyi Biotec GmbH. Milliplex® MAP Kit, Mouse Cytokine/Chemokine Magnetic Bead Panel, was purchased from EMD Millipore Corporation Merck KGaA. FluoSpheres®Carboxylate, Yellow-green (505/515) conjugated, was obtained from Life Technologies. CFSE (5-(and6)-Carboxyl-fluorescein diacetate succinimidyl eyter, CFDA SE) and CD274 (B7-H1) Functional Grade Purified antibody were purchased from eBioscience.
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!