Surface and intracellular staining for flow cytometry analysis were performed as described previously (23 (link), 26 (link)). The antibodies used for surface staining included APC-Cy7-anti-CD4, Pacific Blue-anti-CD8a, APC-anti-CD44, APC-anti -PD-1, PerCP-Cy5.5-anti-CD43, PE-Cy7-anti-CD62L, PE-anti-CTLA-4, PE-anti-LAG-3 (all from BD Biosciences), PE-anti-CD45.1, PE-anti-CD45.2, and PE-Cy7-anti-CD45.2 (all from BioLegend). For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen) and were stained with APC-anti-interferon (IFN)γ, PerCP-Cy5.5-anti-interleukin (IL)-2, FITC-anti-tumor necrosis factor (TNFα) (from BD Bioscience). For Ki67 staining, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (Biolegend) and were stained with BV421-anti-Ki-67 (Biolegend). Freshly isolated cells were used for all assays and about 20,000–40,000 T cells were acquired for each sample using BD FACSCanto II flow cytometer. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506 (eBioscience).
Anti cd45 pe
Manufactured by BioLegend
Sourced in United States, United Kingdom
Anti-CD45-PE is a fluorescently-labeled monoclonal antibody that binds to the CD45 antigen expressed on the surface of hematopoietic cells. It can be used in flow cytometry applications to identify and analyze various leukocyte populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd8 apc, by BioLegend (4 mentions)
Anti cd3 fitc, by BioLegend (4 mentions)
Anti cd4 fitc, by BioLegend (4 mentions)
Anti cd11b apc, by BioLegend (4 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Anti cd34 pe, by BioLegend (3 mentions)
Anti cd44 pe, by BioLegend (3 mentions)
Intracellular fixation and permeabilization buffer set, by Thermo Fisher Scientific (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
Anti cd4 apc cy7, by BD (2 mentions)
Anti cd44 apc, by BD (2 mentions)
Pe anti ctla 4, by BD (2 mentions)
Anti pd 1 apc, by BD (2 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (2 mentions)
Anti cd86 pe, by BioLegend (2 mentions)
Anti cd90 pe, by BioLegend (2 mentions)
Anti cd206 pe, by BioLegend (2 mentions)
Anti cd3 apc, by BioLegend (2 mentions)
Anti c kit igg allophycocyanin apc secondary antibody, by BioLegend (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
33 protocols using anti cd45 pe
1
Multiparametric Flow Cytometry Analysis
2
Multiparametric Flow Cytometry of EVs, Cells
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For flow cytometry of EVs, the isolated EVs were prebound to aldehyde/sulfate latex beads (4 μm) before antibody staining. For flow cytometry of pulmonary immune cells, lung tissues were digested in a solution composed of 10 mM HEPES, 10 μg mL -1 DNAse I and 0.4 mg mL -1 collagenase D and 2% FBS for 30 min. For flow cytometry of peripheral blood, red blood cells (RBCs) were lysed using RBC lysis buffer (Biolegend). For antibody staining, anti-CD29-Alexa Fluor 488, anti-CD44-APC, anti-CD45-PE, anti-CD90-FITC, anti-CD117-APC, anti-Sca-1-APC, anti-CD63-PE, anti-CD81-PE, anti-CD11b-APC, anti-Ly6G-Alexa Fluor 488, anti-Ly6C-PE, and anti-CD45-PE (Biolegend or eBioscience) were used. Finally, the stained cells were analyzed by flow cytometry (Millipore Guava easyCyte) and data were analyzed using the FlowJo software.
3
Flow Cytometry Antibody Panel for Immune Cell Analysis
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Antibodies used for flow cytometry include the following: FITC–anti-CD19 (rat IgG2a; 1:200, #115506; BioLegend), PE–anti-CD45.1 (mouse [A.SW] IgG2a; 1:100, #110707; BioLegend), PE/Cy7–anti-Thy1.1 (mouse IgG1; 1:100, #202518; BioLegend), BV421–anti-CD4 (rat IgG2b; 1:150, #100438; BioLegend), APC–anti-CD45.2 (mouse [SJL] IgG2a; 1:100, #109813; BioLegend), V500–anti-CD3e (Syrian hamster IgG2; 1:50, #560771; BD Biosciences), FITC–anti-CD25 (rat IgG2b; 1:100, #101907; BioLegend), PerCP/Cy5.5–anti-CD19 (rat IgG2a; 1:100, #152405, BioLegend), PerCP/Cy5.5–anti-NK1.1 (mouse IgG2a; 1:100, #108727; BioLegend), PerCP/Cy5.5–anti-CD8b (rat IgG2b; 1:100, #126609; BioLegend), BV605–anti-CD44 (rat IgG2b; 1:100, #103047; BioLegend), FITC–anti-CD8a (rat IgG2a; 1:100, #100705; BioLegend), BV510–anti-CD11b (rat IgG2b; 1:100, #101245; BioLegend), BV421–anti-Ly6C (RçAT IgM; 1:100, #562727; BD Biosciences), and PerCP/Cy5.5–anti-Ly6G (rat IgG2a; 1:100, #127615; BioLegend). For mass cytometry experiments, metal-tagged antibodies were obtained from Fluidigm or conjugated using the Maxpar X8 Antibody Labeling kit according to the manufacturer’s instructions (Fluidigm). Clone and tag information can be found in Table S1.
4
Phenotypic Analysis of Immune Cell Subsets
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Cells were suspended in MACS buffer and incubated with anti-CD16/32 mAb (BioLegend, 101310) for Fc receptor blocking. Cells were stained with the following fluorochrome-conjugated antibodies: PerCP/Cy5.5 anti-CD45 (BioLegend, 103131), PE anti-CD45 (BioLegend, 109807), FITC anti-CD8 (MBL, D271-4), APC anti-CD8 (BioLegend, 100712), PE anti-GzmB (BD, 561142), PerCP/Cy5.5 anti-CD11c (BioLegend, 117328), PE/Cy7 anti-MHC II (BioLegend, 107630), PE anti-CD86 (BioLegend, 105008), APC anti-CD80 (BioLegend, 104713), FITC anti-CD40 (BioLegend, 102905), PE anti-ITGα5 (BioLegend, 103805), anti-ITGα4 (BioLegend, 103607), anti-ITGαv (BioLegend, 104105), anti-ITGβ1 (BioLegend, 102207), anti-ITGβ2 (BioLegend, 101407), anti-ITGβ3 (BioLegend, 104307), and anti-PIR-A/B (BD, 550349). For analysis of OVA-specific CD8+ T cells, cells were incubated with PE-conjugated H-2Kb-SIINFEKL-tetramer (MBL), according to the manufacturer's instructions. Stained cells were analyzed by BD FACSVerse (BD bioscience). Data analysis was performed using FlowJo software (Treestar).
5
Flow Cytometric Analysis of Cell Surface Markers
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Cells were incubated with a specific monoclonal antibody conjugated with phycoerythrin (PE) in 200 μl PBS for 30 min in the dark at 4°C and then analyzed by flow cytometry. Antibodies against PE anti-CD11b/c (201807; BioLegend), PE anti-CD29 (102207; BioLegend), PE anti-CD45 (202207; BioLegend), and PE anti-CD90 (202523; BioLegend) were used. PE anti-mouse IgG1 (4066607; BioLegend) was used as the control.
6
Isolation and Characterization of Rat BMSCs
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BMSCs were isolated and cultured from the healthy male SD rats weighing 80–100 g as described previously [35 (link)]. Cells at 3rd to 5th passage (P3 to P5) were utilized for subsequent experiments. The morphology of the BMSCs was observed under a common optical microscope. The P3 BMSCs were identified by flow cytometry and the phenotype was characterized with the following antibodies: PE-Cy7-anti-CD29, Alexa Fluor 488-anti-CD90, Alexa Fluor 647-anti-CD11b, and PE-anti-CD45 (BioLegend, San Diego, CA, USA). BMSCs were used as the EVs donor cells in the study.
7
Multicolor Flow Cytometry Immunophenotyping
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Cells of interest were stained with different combinations of the following antibodies: FITC anti-CD3, brilliant violet 510 (BV510) anti-CD4, peridinin chlorophyll (PerCP)-conjugated anti-CD4, APC anti-CD8, APC/Cy7 anti-CD8, APC/Cy7 anti-CD45, PE anti-CD45, PE anti-CD45RO, APC/Cy7 anti-CD62L (all BioLegend) and PE-conjugated HLA-A*02:01/CMVpp65p-specific dextramer (Immudex, Copenhagen, Denmark). Surface staining was performed at room temperature for 20 min in the dark and washed with PBS (Lonza, Vervies, Belgium) with 0.1% human AB serum (c.c. pro). 7-amino-actinomycin D (7-AAD; BD Biosciences) was applied prior to flow cytometric analysis to exclude dead cells. All samples were analyzed by multicolor flow cytometry (FACS Canto II, FACSDiva V8.1.2 software, FlowJo_v10.7.1 software; all BD Biosciences). Gates were set based on the forward scatter versus side scatter properties of lymphocytes. At least 30,000 events were acquired in the CD3+ gate.
8
Multiparametric Analysis of Mouse T Cells
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Isolated mouse T cells were TCR-stimulated as previously described for 1–3 d. For GPI-anchored proteins, antibodies included PE-anti-CD45.2, FITC-anti-Sca-1, PE-anti-CD109, APC-anti-CD24, PE/Cy5-anti-CD44, APC-anti-CD90, PE-anti-CD59a, PE/Cy7-anti-CD37, and PE/Cy5-anti-CD48 (all from BioLegend). For PE staining, cells (0.5 × 106) were fixed with 200 μL of fixation buffer (BioLegend) in a 96-well plate in the dark for 20 min at room temperature, then centrifuged at 400×g for 5 min and the supernatant was discarded. The cells were resuspended and permeabilized in 250 μL of Intracellular Staining Perm Wash Buffer (BioLegend) and centrifuged at 400×g for 10 min. The permeabilization steps were repeated twice. The cells were resuspended and stained in 100 μL of Perm Wash Buffer with 0.5 μM of duramycin-LC-fluorescein or duramycin-LC-Cy5 (Molecular Targeting Technologies). As a control, 1 μM of BSA-FITC (Nanocs, Inc.) was added to the cells for 30 min in the dark at room temperature. After staining, the cells were washed twice with 250 μL of Perm Wash Buffer or TES buffer and centrifuged at 400×g for 5 min. The cells were then resuspended in 150 μL of Cell Staining Buffer (BioLegend) and analyzed together with non-stained controls using a BD LSRFortessa cell analyzer, and the data were analyzed using FlowJo software (BD Biosciences).
9
Antibody-based Western Blot and Flow Cytometry
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Antibodies for Western blots included anti-MEK1, anti-ERK1/2, and anti-p-ERK1/2 (catalog no.: 12671S, 1:1000 dilution; catalog no.: 9107S, 1:1000 dilution; catalog no.: 4370T, 1:500 dilution; Cell Signaling); anti-FLAG (catalog no.: F1804; 1:1000 dilution; Millipore Sigma), anti-GAPDH (catalog no.: ab9485; 1:1000 dilution; Abcam), anti-p-Elk1 (catalog no.: 43004; 1:1000 dilution; QED Bioscience), anti-Elk1 and anti-β-actin (catalog no.: SC-365876, 1:1000 dilution and SC-517582, 1:2000 dilution, respectively; Santa Cruz). BioLegend antibodies for flow cytometry used at manufacturer's recommended concentrations included FITC-anti-CD3 (catalog no.: 100306), APC-anti-CD8 (catalog no.: 100712), and PE-anti-CD45.2 (catalog no.: 109808). Dead cells were detected with BV421 viable dye (Invitrogen). Mimic Dharmacon miRIDIAN reagents for miR-15a and miR-16 (catalog nos.: CTM-535609 and CTM-535610) as well as negative control mimic (catalog no.: CTM-535611) were purchased with 5′-fluorescein and 3′-cholesterol modifications. DOX was purchased from Sigma.
10
Hypoxia-Induced BMSC Viability Assessment
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Total bone marrow cells were flushed from the femurs and tibias of rats (2- to 4-month-old) with culture medium; the rats were sacrificed via sevoflurane overdose as previously described [31 (link)]. Complete L-DMEM containing 15% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin was used to resuspend the BMSCs, and then, the BMSCs were incubated in a humid chamber. At 48 h, the first medium change was performed to remove the nonadherent cells. When the cells reached 90% confluence, they were harvested with 0.25% trypsin (Sigma) and passaged at a ratio of 1 : 2. FCM was performed to assess the BMSC surface markers. The cells were incubated with the following fluorochrome-conjugated primary antibodies (all from BioLegend, USA): anti-CD90-PE, anti-CD29-APC, and anti-CD45-PE. BMSCs from passage (P) 3 to P5 were used for subsequent experiments.
The cells were stimulated with hypoxia, and cell viability was detected [32 (link)]. Approximately 5 × 105 BMSCs suspended in L-DMEM were plated in 150 mm-diameter culture dishes. The cells were then separately cultured under the conditions below for 12, 24, 48, 72, 96, or 120 h: 10% exosome-free FBS with hypoxia (94% N2, 5% CO2, and 1% O2 gas mixture). BMSC viability was analyzed with CCK-8 assays. Briefly, adherent cells were digested with 0.05% trypsin and collected for CCK-8 assay according to the manufacturer's instructions.
The cells were stimulated with hypoxia, and cell viability was detected [32 (link)]. Approximately 5 × 105 BMSCs suspended in L-DMEM were plated in 150 mm-diameter culture dishes. The cells were then separately cultured under the conditions below for 12, 24, 48, 72, 96, or 120 h: 10% exosome-free FBS with hypoxia (94% N2, 5% CO2, and 1% O2 gas mixture). BMSC viability was analyzed with CCK-8 assays. Briefly, adherent cells were digested with 0.05% trypsin and collected for CCK-8 assay according to the manufacturer's instructions.
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