The DMEM medium, PBS medium, and 10% fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, NY, USA). The tumor dissociation kit and spleen dissociation kit were obtained from Miltenyi Biotec (San Diego, CA, USA). Anti-mouse PerCP/Cy5.5 CD3, anti-mouse FITC CD4, anti-mouse PE CD8, anti-mouse APC CD25, anti-mouse FITC Gr-1, anti-mouse PE CD11b, anti-mouse PerCP/Cy5.5 Ly6c, and anti-mouse APC Ly6g were all purchased from Biolegend (San Diego, CA, USA). 5%BSA, paraformaldehyde, electrophoresis solution, transfer solution, CCK-8 kit, RIPA were all provided by Beyotime Biotech (Beijing, China). The SDS-PAGs (Sodium Dodecyl Sulfate-Polyacrylamide Gels) were bought from Dakewe Biotech (Shenzhen, China). RT-PCR primers were designed and provided by Sangon Biotech (Shanghai, China). Trizol reagent, Tween-20, and 20 × TBS buffer were obtained from Thermo Scientific (Rockford, IL). Antibodies for Western blot were all purchased from Cell Signaling Technology (MA, USA). An ECL Kit was provided by Tanon Biotech (Shanghai, China).
Apc anti mouse cd25
Manufactured by BioLegend
Sourced in United States
APC anti-mouse CD25 is a fluorescence-labeled antibody that binds to the CD25 cell surface marker on mouse cells. CD25 is the alpha chain of the interleukin-2 receptor and is expressed on activated T cells and regulatory T cells. The APC fluorophore allows for detection of CD25-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Fitc anti mouse cd4, by BioLegend (7 mentions)
Pe anti mouse foxp3, by BioLegend (4 mentions)
Cck 8 kit, by Beyotime (2 mentions)
Tween 20, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Beyotime (2 mentions)
Electrophoresis solution, by Beyotime (2 mentions)
Transfer solution, by Beyotime (2 mentions)
Anti mouse percp cy5.5 ly6c, by BioLegend (2 mentions)
Tbs buffer, by Thermo Fisher Scientific (2 mentions)
Apc anti mouse ly6g, by BioLegend (2 mentions)
Pe anti mouse cd11b, by BioLegend (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Anti mouse fitc gr1, by BioLegend (2 mentions)
Rt pcr primers, by Sangon (2 mentions)
Pe anti mouse cd8a, by BioLegend (2 mentions)
Pe anti mouse human cd11b, by BioLegend (2 mentions)
Anti mouse cd3ε pe cy7, by BioLegend (2 mentions)
12 protocols using apc anti mouse cd25
1
Tumor and Spleen Cell Isolation Protocol
2
Analyzing Gemcitabine's Apoptotic Effects
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The DMEM medium and 10% FBS were obtained from Gibco Life Technologies (Grand Island, NY, USA). Gemcitabine (GEM) was brought from Selleckchem. The PBS medium, Annexin V-FITC/PI apoptosis detection kit, and BCA protein assay kit were purchased from KeyGEN Biotech (Jiangsu, China). 5%BSA, paraformaldehyde (PFA), electrophoresis solution, transfer solution, CCK-8 kit, RIPA, and SDS-PAGE gel were all provided by Beyotime Biotech (Beijing, China). The mouse tumor and spleen dissociation kits were obtained from Miltenyi (MACS, Miltenyi Biotec, Germany). Anti-mouse PE-CD11b, anti-mouse FITC-Gr-1, anti-mouse PerCP/Cy5.5-Ly6C, anti-mouse APC-Ly6G, anti-mouse PE NKG2D, anti-mouse APC-CD49b, anti-mouse FITC-CD4, and anti-mouse APC-CD25 were purchased from BioLegend. RT-PCR primers were designed and provided by Sangon Biotech (Shanghai, China). Trizol reagent, Tween-20, and 20× TBS buffer were obtained from Thermo Scientific (Rockford, IL). An ECL Kit was provided by Tanon Biotech (Shanghai, China).
3
Murine Immune Cell Phenotyping
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Single-cell suspensions were prepared from the spleens and peripheral blood of mice. Spleens were mechanically disrupted using the plunger end of a 10 ml syringe and resuspended in 1% FBS/PBS. Peripheral blood in centrifuge tubes was placed into a 4℃ high speed centrifuge and spun at 1500 rpm for 5 min. The supernatant was frozen at -20℃ for subsequent ELISA detection. Then, 2 ml of erythrocyte lysate was added to the precipitate of the centrifuge tube, vibrated and incubated at room temperature for 10 min. Then, the tubes were centrifuged, the supernatant was discarded, and the cells were resuspended in 1% FBS/PBS.
Zombie NIR Fixable Viability Kit (Biolegend, USA) were used to stain dead cells. For cell-surface markers, single-cell suspensions were harvested and incubated with anti-mouse/human CD11b-PE, anti-mouse Ly-6G/Ly-6C (Gr-1)-APC, anti-mouse CD4-FITC, anti-mouse CD8a-PE, anti-mouse CD25-APC, anti-mouse FOXP3-PE or appropriate isotype controls (Biolegend, USA) for 30 min at 4 °C. Fixation buffer was added for 20 min, then cells were resuspended in 1% PBS. Finally, all samples were analysed by flow cytometry, and data analysis was processed using FlowJo V10 (Treestar, Inc.).
Zombie NIR Fixable Viability Kit (Biolegend, USA) were used to stain dead cells. For cell-surface markers, single-cell suspensions were harvested and incubated with anti-mouse/human CD11b-PE, anti-mouse Ly-6G/Ly-6C (Gr-1)-APC, anti-mouse CD4-FITC, anti-mouse CD8a-PE, anti-mouse CD25-APC, anti-mouse FOXP3-PE or appropriate isotype controls (Biolegend, USA) for 30 min at 4 °C. Fixation buffer was added for 20 min, then cells were resuspended in 1% PBS. Finally, all samples were analysed by flow cytometry, and data analysis was processed using FlowJo V10 (Treestar, Inc.).
4
Foxp3+ Regulatory T Cell Isolation
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The splenocytes were isolated from spleens of experimental mice and cultured in RPMI 1640 medium (Thermo Fisher Scientific Inc.). The cell surfaces were blocked with rat anti-mouse CD16/32 antibody for 15 min at 4°C and then incubated with anti-mouse CD3ε-Pecy7, anti-mouse CD4-FITC, and anti-mouse CD25-APC (Biolegend, United States) for 30 min at 4°C in the dark. After being washed and resuspended in 300 μL fixation buffer (Thermo Fisher Scientific Inc.) for 30 min at 4°C and then in 1 mL permeabilization buffer (Thermo Fisher Scientific Inc.) for 30 min at 4°C, the cells were stained with anti-mouse Foxp3-PE (Biolegend) for 30 min at 4°C in the dark. The isotype-matched immunoglobulins (Biolegend, London, United Kingdom) and fluorescence minus one were used as controls for non-specific staining as baseline. The cells were washed three times and resuspended in 400 μL of 2% paraformaldehyde and detected by DxP AthenaTM flow cytometer (CYTEK, United States). All the data were analyzed using FlowJo-V10 software (BD Biosciences, United States).
5
Treg Analysis in Tumor Samples
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For Treg analysis, tumours were digested using a gentleMACS Dissociator and Tumour dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), while red blood cells were lysed with 1 × RBC Lysis Buffer (BioLegend, San Diego, CA, USA). Per sample, 2 × 106 cells in the FCM buffer (PBS containing 5% FBS and 2 mM EDTA) were treated with anti-mouse CD16/32 antibodies (RRID:AB_467134 , 14-0161-85; ThermoFisher Scientific) for 30 min at 4 °C and stained with anti-mouse CD45 PE-Cy7 (RRID:AB_2734986 , 25-0451-82; ThermoFisher Scientific), anti-mouse CD4 PE (RRID:AB_2621736 , 50-0041; Cytek Biosciences, Fremont, CA, USA), and anti-mouse CD25 APC (RRID:AB_312861 , 102012; BioLegend) for 30 min at 4 °C. Cells were subsequently washed twice with the FCM buffer, stained with 7-aminoactinomycin D (BioLegend), and analysed on an Attune NxT Flow Cytometer (ThermoFisher Scientific). FCM data were analysed using FlowJo software (Becton, Dickinson and Company, Ashland, OR, USA). Tumour growth data were statistically evaluated using the Wilcoxon rank sum test in the GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA).
6
Baicalein Protocol for Immune Cell Analysis
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Baicalein (BA, C15H10O5, molecular weight: 270.24, purity ≥99%, Figure 1A ) was purchased from Shanghai Ronghe Co. (Shanghai, China), and was dissolved in dimethyl sulfoxide (DMSO) and kept in −80°C. The following antibodies were obtained from Biolegend (San Diego, CA, United States): anti-mouse CD206-AF647 (Cat#141712), anti-mouse Foxp3-PE (Cat#320008), anti-mouse CD4-PerCP/Cy5.5 (Cat#100434), anti-mouse CD11b-FITC (Cat#101206), anti-mouse CD25-APC (Cat#101910), anti-mouse CD45-APC/Cy7 (Cat#157617), anti-mouse CD11c-PE/Cy7 (Cat#117318), anti-mouse F4/80-PE (Cat#123110), anti-mouse CD8a-FITC (Cat#155004), and anti-mouse CD3ε-PE/Cy7 (Cat#155706). Fixable Viability Stain (FVS) 700 was purchased from BD Bioscience (San Jose, CA, United States, Cat#564997). IFN-γ was supplied by Peprotech (Cranbury, NJ, United States, Cat#500-M90). Phorbol 12-myristate 13-acetate (PMA) was purchased from Lianke (Hangzhou, China, Cat#70-CS0001). Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (Merck Life Science, Darmstadt, Germany, SKU#L3129-25 MG).
7
Comprehensive Immune Profiling of Murine Tumors
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Carprofen (Rimadyl® Injectable, 50 mg/mL) was purchased from Patterson Companies (Saint Paul, MN, USA). Pacific Blue™ anti-mouse CD3, FITC anti-mouse CD4, PerCP anti-mouse CD8a, PE anti-mouse/human CD11b, APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1), PE/Cy7 anti-mouse CD62L, APC anti-mouse/human CD44, APC anti-mouse CD25, Biolegend PE anti-mouse/rat/human FOXP3, PerCP anti-mouse CD11c, PE/Cy7 anti-mouse CD86, FITC anti-mouse I-A/I-E, and APC anti-mouse CD40 were purchased from Biolegend (San Diego, CA, USA). Mouse tumor dissociation kit (No. 130-096-730) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).
8
Thymus and Spleen Cell Isolation
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The thymus and spleen were dissected from mice and then ground in cold PBS. The cell suspension was filtered through a 40 μm strainer. Red blood cells were lysed with red blood cell lysis buffer (70 (link)). The rest of the cells were analyzed by FACS for cell percentages or for cell culture. For thymocyte subpopulation assays, the cells were stained with the following surface antibodies in flow cytometry staining buffer (486.5 ml of 1× PBS, 12.5 ml of goat serum, and 1 ml of 0.5 M EDTA): PE anti-mouse CD127 (BioLegend), APC anti-mouse CD25 (BioLegend), FITC anti-mouse CD4 (BioLegend), PE-Cy5.5 anti-mouse CD8 (BioLegend), PE-Cy7 anti-mouse CD3 (BioLegend), and APC-Cy7 anti-mouse CD45 (BioLegend). The cells were incubated with the antibodies above in the tubes on ice for 45 min. The different subpopulations were counted and collected by FACS sorting (BD FACSAria III).
9
Immune Cell Changes After Brain Injury
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To evaluate the changes of immune cells in the wound and blood, we collected the scalp and blood in different groups on day 6 post brain injuries. The fresh wound tissues were digested with collagenase and hyaluronidase (Stemcell, 07919) overnight at 37 °C to obtain single cells for flow cytometry analysis (BD FACSCanto II). Lymphocytes in the blood were collected with Ficoll kits (Solarbio, P8620). Lymphocytes were stained with PE anti-mouse F4/80 (Biolegend, 123110), APC anti-mouse CD80 (Biolegend, 104714), FITC anti-mouse CD206 (MMR) (Biolegend, 141704), APC/Cyanine7 anti-mouse CD3 (Biolegend, 100222), PE/Cyanine7 anti-mouse CD8a (Biolegend, 100722), FITC anti-mouse CD4 (Biolegend, 100406), PE anti-mouse NK-1.1 (Biolegend, 156504), PerCp anti-mouse CD45 (Biolegend, 103130), APC anti-mouse CD19 (Biolegend, 152410), APC anti-mouse CD25 (Biolegend, 101909), and PE-Foxp3 (FJK-16s) (Thermo, 12-5773-82) antibodies, PE Rat IgG2a, ĸ Isotype Ctrl Antibody (Biolegend, 400508), FOXP3 Monoclonal Antibody (FJK-16s), PE (eBioscienceTM InvitrogenTM, 12-5773-82).
10
Multiparametric Flow Cytometry Analysis of Mouse Immune Cells
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100 μL of peripheral blood sample was lysed by RBC Lysis Buffer, cells were collected and suspended in Cell Staining Buffer (BioLegend, Cat# 420201). Flow cytometry was performed performed on FACS Aria III (Becton Dickinson) using standard methods. For detection of surface antigens, cells were washed and stained with saturating amounts of antibodies conjugated with fluorescein in the presence of FcR-specific blocking antibody for 20 min on ice. Antibodies used were as follows: Alexa Fluor-488 anti-mouse Gr-1 (Biolegend, Cat# 108417, clone: RB6-8C5, 1:100), PE anti-mouse CD19 (Biolegend, Cat# 152408, clone: 1D3/CD19, 1:100), Alexa Fluor-647 anti-mouse CD3ε (Biolegend, Cat# 100322, clone: 145-2C11, 1:100), PE anti-mouse CD8a (Biolegend, Cat# 100708, clone: 53-6.7, 1:100), Alexa Fluor-488 anti-mouse CD4 (Biolegend, Cat# 100423, clone: GK1.5, 1:100), Alexa Fluor-647 anti-mouse c-Kit (Biolegend, Cat# 105818, clone: 2B8, 1:100), APC anti-mouse CD25 (Biolegend, Cat# 102012, clone: PC61, 1:100), PE/Cy5 anti-mouse/human CD44 (Biolegend, Cat# 103010, clone: PC61, 1:100), Alexa Fluor-647 anti-mouse/human Foxp3 (Biolegend, Cat#320013, clone: 150D, 1:100), PE anti-mouse/human CCR4 (Biolegend, Cat# 131203, clone: 2G12, 1:100). The data were analyzed by FlowJo_V10 software. The gating strategy are shown in Supplementary Fig. 6 .
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