CD4+CD25- or CD4+ T cells were isolated after mechanical disruption of murine spleens by using the CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec) or CD4+ BD IMag beads, respectively, via manufacturer’s instructions. B cells were isolated from murine spleens after mechanical disruption by enriching for CD19+ B cells labeling with CD19+ magnetic beads and processing with MACs columns (Miltenyi Biotec). To isolate DCs, spleens were treated with collagenase and processed using a spleen dissociation kit (Miltenyi Biotec) and subsequently labeled with CD11c+ magnetic beads (Miltenyi Biotec). DCs or B cells were pulsed with 1 or 2 μg/ml of CBirTox for 1–2 hours, respectively, at 37°C. After washing with PBS, DCs and B cells were cultured with CD4+CD25- or CD4+ CBir1 Tg T cells at a ratio of 1:10 or 2:1, respectively with or without the addition of anti-TGF-β (1,2,3) (1D11) (R&D Systems), LE135, retinoic acid (Tocris), or TGF-β R1 kinase inhibitor III (Calbiochem) in complete 1640 RPMI with L-gluatmaine media, supplemented with penicillin (1U/ml), streptomycin (100 μg/ml), 2.5 μM 2-ME and 10% fetal bovine serum (FBS) (Atlanta Biologicals).
Spleen dissociation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The Spleen Dissociation Kit is a laboratory equipment designed for the isolation and separation of cells from spleen tissue. It provides a streamlined process for mechanical and enzymatic dissociation of spleen samples, enabling the recovery of viable single-cell suspensions for further analysis or experimentation.
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Lab products found in correlation
Cd11c microbeads ultrapure, by Miltenyi Biotec (3 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Dnase 1, by Roche (2 mentions)
100 m cell strainer, by BD (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Retinoic acid, by Bio-Techne (1 mentions)
Le135, by Bio-Techne (1 mentions)
Cd19 magnetic bead, by Miltenyi Biotec (1 mentions)
Anti tgf β 1 2 3 1d11, by R&D Systems (1 mentions)
Macs column, by Miltenyi Biotec (1 mentions)
Cd11c magnetic beads, by Miltenyi Biotec (1 mentions)
Gentlemacs octo dissociator with heater, by Miltenyi Biotec (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
20 protocols using spleen dissociation kit
1
Isolation and Co-culture of Murine Immune Cells
2
Isolating pDCs and Pan T Cells
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For pDCs isolation, the spleens were dissociated into single-cell suspensions with spleen dissociation kit (130-095-926, Miltenyi Biotec, Bergisch Gladbach, Germany) on the gentleMACS Octo Dissociator with Heaters (130-096-427, Miltenyi Biotec, Bergisch Gladbach, Germany) running program 37C_m_SDK_1. For pan T cells isolation, the spleen single-cell suspensions were obtained by grinding followed by filtration through a nylon mesh. pDCs and pan T cells were purified from splenocytes using pDCs isolation kit (130-107-093, Miltenyi Biotec, Bergisch Gladbach, Germany) and pan T cell isolation kit II (130-095-130, Miltenyi Biotec, Bergisch Gladbach, Germany) respectively, according to the manufacturer’s instructions.
3
Activated Splenocytes Cytotoxicity Assay
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In order to obtain the activated splenocytes as effector cell, 1×106 4T1 cells were orthotopically implanted into the mammary fat pad of female BALB/c mice on day 0. The average volume of tumors was approximately 100 mm3 on day 8, the mice were divided into 4 groups randomly to receive the following treatments: Hepes, free DOX&IMQ, LT-DOX, LT-DOX+LT-IMQ. The dosages of DOX and IMQ were 3 and 0.75 mg/kg, respectively. The second treatment with the same dosing regimens was given to each group on day 11. Three days later, the mice were sacrificed and the spleens were harvested. The spleen dissociation kit (Miltenyi Biotec Germany) were utilized to prepare the solution of splenocytes, which were further incubated with mitomycin C-treated 4T1 tumor cells for 72 h at 37 °C under 5% CO2 with mouse IL-2 added into the culture media. After stimulation, viable splenocytes were used as effector cells for the measurement of specific cytotoxicity activity. And 4T1 cells were used as target cells. The splenocytes obtained from LT-DOX+LT-IMQ group were divided into two parts, one of which was added to 4T1 cells that had been given anti-PD-L1 in advance, while the other was added to normal 4T1 cells. The cytotoxicity of cytotoxic T lymphocytes was determined by LDH kits in accordance with manufacturer׳s protocol.
4
Calcium Mobilization Assay with P2RX7
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20.103 HEK293T-mP2RX7C57BL/6J or HEK293T-pcDNA6 cells were seeded per well on a poly-L-Lysine (Sigma-Aldrich) 96 well-coated plate in complete medium. Twenty-four hours later, cells were washed in sucrose buffer (300 mM sucrose, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM Glucose, 20 mM HEPES, pH 7-7.4) and incubated for 1 h at 37 °C with 1 µM of Fluo-4 AM (Life Technologies). Cells were washed once with PBS + 5% FBS then twice with the sucrose buffer. Fluo-4 AM fluorescence was read on a Xenius, microplate reader (SAFAS) at 485/528 nm at 37 °C. After 3 min of baseline readings, 333 µM of ATP (Sigma-Aldrich) were added with or without various concentrations of HEI3090. For the assay on splenocytes, spleens were digested with the spleen dissociation kit (Miltenyi Biotech). 5.105 splenocytes were seeded per well on a 96-well plate in sucrose buffer and incubated for 30 min at RT with 1 µM of Fluo-4-AM.
5
Spleen Immune Cell Analysis
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Spleens from 7-wk-old male control mice and M98A/M98A mice were treated with the Spleen Dissociation Kit (Miltenyi). Red blood cells were lysed with ACK Lysis Buffer (Life Technologies). Splenocytes were stained with mAbs for surface molecules and then with Zombie UV Viability Dye. Intracellular CD74 and H2-DM were stained using the FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). After fixation, cells were stained with anti-CD74 antibody and anti–H2-DM antibody, washed with permeabilization buffer, and stained with FITC-conjugated anti-rat IgG1 secondary antibody.
6
Isolation and Purification of Splenic Dendritic Cells
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Spleen was collected and dissociated enzymatically using a spleen dissociation kit (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). The spleen was then mechanically dissociated using 40 μm nylon cell EASYstrainer (Greiner bio-one, Dutscher, Brumath, France). Splenic DCs were purified using a positive selection kit (CD11c Microbeads Ultrapure, Miltenyi Biotec GmbH) following the manufacturer’s instructions. Splenic DCs were then maintained in the culture medium at a concentration of 1 × 106 to 2 × 106 cells/mL. The splenic DCs’ purity was assessed by flow cytometry after antibody stanning, as described below, with anti-CD11c-PE-Vio®770 antibody (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). With this method, the degree of purity was between 85% and 95%.
7
Isolation and Activation of Mouse T Cell Subsets
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All procedures were performed according to the protocol provided by Miltenyi Biotec. The spleen was dissociated using a Spleen Dissociation Kit (Miltenyi Biotech) and homogenized using the Miltenyi GentleMACS system. Splenocytes were further processed using the mouse CD8a+ T Cell Isolation Kit and the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech). Isolated cells were cultured at a density of 1.5 × 106/mL in complete RPMI media with recombinant IL-2 (Sigma-Aldrich, St. Louis, Missouri) at 2500 U/mL. Cells were seeded at a density of 1.5 × 106/mL in a 24-well flat-bottom plate. Isolated cells were activated overnight with Gibco Dynabeads Mouse T-Activator CD3/CD28 for T Cell Expansion and Activation (Thermo Fisher Scientific, Inc.). Cytotoxic T cells were activated at a T cell:bead ratio of 1:1 and Treg cells at a Treg:bead ratio of 1:2.
8
Adoptive Transfer of Splenocytes in Murine Pulmonary Fibrosis
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Spleens from C57BL/6J male mice (8–10 weeks) were collected and digested with the spleen dissociation kit (Miltenyi Biotech) according to the supplier’s instructions. 3 × 106 splenocytes were injected i.v. in P2rx7−/− mice 1 day before intranasal delivery of BLM or when indicated in repeated administration. Mice were treated i.p. every day for 14 days with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO). Nlrp3−/− spleens were a kind gift from Dr Laurent Boyer, Il18−/− spleens from Dr George Birchenough and Il1b−/− spleens from Dr Bernhard Ryffel, all on a C57BL/6J background.
9
Evaluating Tumor-Infiltrating Immune Cells
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Six days after tumor implantation, animals with an average tumor volume of 80–100 mm3 were selected and divided into 5 groups randomly (n = 3). Mice of each group received 3 i.v. injections, and were sacrificed 7 days after the last dose and their spleens were collected. Splenocytes suspensions were prepared by using the Spleen Dissociation kit (Miltenyi Biotec Germany). The extracted spleen cells were stained with anti-CD11c-PE, anti-CD86-FITC and anti-CD80-FITC, and then detected by flow cytometry.
To analyze the CD8+ and CD4+ T cell responses in tumors, tumors were harvested from mice in different groups and stained with anti-CD3e-eFluor 610, anti-CD8a-FITC, anti-CD4-FITC antibodies according to the manufacturer׳s protocols. Briefly, tumor tissues were cut into small pieces and put into a glass homogenizer containing PBS (pH 7.4) with 2% heat-inactivated fetal bovine serum. Then, the single-cell suspension was prepared with the homogenizer without addition of digestive enzyme. Finally, cells were stained with fluorescence-labelled antibodies after the removal of red blood cells (RBC) using the RBC lysis buffer.
Serum samples were isolated from mice after various treatments and diluted for analysis. Tumor necrosis factor (TNF-α) and interferon gamma (IFN-γ) were analyzed with ELISA kits according to the protocols of vendors.
To analyze the CD8+ and CD4+ T cell responses in tumors, tumors were harvested from mice in different groups and stained with anti-CD3e-eFluor 610, anti-CD8a-FITC, anti-CD4-FITC antibodies according to the manufacturer׳s protocols. Briefly, tumor tissues were cut into small pieces and put into a glass homogenizer containing PBS (pH 7.4) with 2% heat-inactivated fetal bovine serum. Then, the single-cell suspension was prepared with the homogenizer without addition of digestive enzyme. Finally, cells were stained with fluorescence-labelled antibodies after the removal of red blood cells (RBC) using the RBC lysis buffer.
Serum samples were isolated from mice after various treatments and diluted for analysis. Tumor necrosis factor (TNF-α) and interferon gamma (IFN-γ) were analyzed with ELISA kits according to the protocols of vendors.
10
Isolation and Characterization of Murine FDCs
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FDCs were isolated from murine spleen samples using a spleen dissociation kit (MiltenyiBiotech, 130-095-926) on a gentileMACS dissociator (MiltenyiBiotec, 130-093-235). Samples were subsequently filtered (70 µm), RBCs lysed using ammonium-chloride-potassium (ACK) buffer and samples depleted for CD45 positive cells, using mouse CD45 MicroBeads (MiltenyiBiotec, 130-052-301) over MACS LS separation columns (MiltenyiBiotec, 130-042-401). Enriched CD45 negative splenocytes were subsequently stained for FDCs using biotinylated FDC-M1 and anti-ICAM-1, then washed and stained with secondary antibodies streptavidin-APC Cy7 and anti-hamster-Alexa561 in conjunction with CD45-BV480, and FcµR-Alexa591 in Brilliant Stain Buffer BD (Biosciences, 563794) (see antibody section for details). CD45 negative, FDC-M1+, ICAM-1+ FDCs were analyzed using BD FACSariaIII (Becton, Dickinson and Company).
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