Mesenteric lymph nodes, cervical lymph nodes, blood, and spleens were collected from the mice, and the samples were cut into small pieces and forced through a 100-μm cell strainer. Single-cell suspensions were stimulated with 40.5 ng⋅mL–1 of phorbol-12-myristate-13-acetate (PMA) and 700 ng⋅mL–1 of ionomycin in the presence of 2.5 μg⋅mL–1 of a protein transport inhibitor (Biolegend, #423303) for 6–8 h at 37°C. Then, the cells were incubated in 5% of fetal calf serum for 15 min and stained with anti-CD45-PerCP (Biolegend, #103130), anti-CD4-FITC (Biolegend, #100510), and anti-IFN-γ-PE/Cy7 (Biolegend, #505825). Aliquots of the cells were fixed and permeabilized with FoxP3/True-NuclearTM Transcription factor buffer set (Biolegend, #424401) and stained with anti-FoxP3-PE (Biolegend, #320007), anti-CD45-PerCP (Biolegend, #103130), and anti-CD4-FITC (Biolegend, #100510).
Anti cd45 percp
Manufactured by BioLegend
Sourced in United States, United Kingdom
Anti-CD45-PerCP is a fluorescently labeled antibody that binds to the CD45 protein, which is expressed on the surface of most hematopoietic cells. The PerCP (peridinin-chlorophyll-protein complex) fluorescent dye is conjugated to the antibody, allowing for its detection in flow cytometry applications.
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Lab products found in correlation
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Anti cd4 fitc, by BioLegend (3 mentions)
Anti cd137 rabbit mab, by Cell Signaling Technology (3 mentions)
Agonistic anti cd137 mab, by BPS Biosciences (3 mentions)
Anti cytokeratin mouse mab, by Abcam (3 mentions)
Anti cd3 fitc, by BioLegend (3 mentions)
Anti cd137 apc, by BioLegend (3 mentions)
Sybr primescript rt pcr kit, by Takara Bio (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
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Mojosort magnet, by BioLegend (3 mentions)
Mojosort human cd8 nanobeads, by BioLegend (3 mentions)
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Anti ifn γ pe cy7, by BioLegend (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Anti foxp3 pe, by BioLegend (2 mentions)
Anti cd11b apc, by BioLegend (2 mentions)
Anti cd11c fitc, by BioLegend (2 mentions)
Mojosort human cd8 cell isolation kit, by BioLegend (2 mentions)
Anti cd8 mouse antibody 66868 1 ig for ihc, by Proteintech (2 mentions)
24 protocols using anti cd45 percp
1
Murine Lymphoid Tissue Analysis
2
Multicolor Flow Cytometry of Leukocyte Activation
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For flow cytometry analysis, 50 μl peripheral venous blood was used to obtain 100,000 leukocytes via the inertial microfluidic system (Fig. 1 ). Leukocytes were incubated for 20 min at RT with the following antibodies to human proteins, with clones noted in parentheses: anti-CD45 PerCP (HI30), anti-CD66b Pacific blue (G10F5), anti-CD16 APC-Cy7 (3G8), anti-CD69 FITC (FN50), pHrodo (PE), anti-CD62L Brilliant Violet 510 (DREG-56), anti-CD42b Alexa Fluor 700 (HIP1) (all from BioLegend) and anti-CD14 APC (61D3) and anti-CD11b PE-Cy7 (ICRF44) (from Thermo Fisher). After staining, the cells were lysed and fixed with 2 ml 1:4 dilution of Lyse/Fix Buffer 5× (BD Phosflow) with distilled water for 15 min at RT. Data were acquired from the BD LSR Fortessa flow cytometer and analysed using FlowJo version 10.1 (Tree Star). PMN (CD45+SSCHiFSCHi) subsets were determined by CD16 and CD66b surface expression. Monocyte (CD45+SSCLowFSCHigh) subsets were determined by CD14 and CD16 surface expression. The molecules of leukocyte activation were assessed, namely the expression of CD62L, CD11b, CD69 and CD42b.
3
T-cell Characterization in Lamina Propria and Lung
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Cells from lamina propria and lungs were stimulated with PMA (Sigma-Aldrich) 50 ng/ml, Ionomycin (Sigma-Aldrich) 500 ng/ml and Brefeldin A (Biolegend, San Diego, CA, USA) 5 µg/ml for 4 h at 37 °C and 5% CO2. T-cell surface staining was performed for 30 min at 4 °C using the following antibodies diluted in PBS: anti-CD45 PercP (BD, Franklin Lakes, NJ, USA), anti-CD4 APCCy7 (Biolegend) and anti-CD8 FITC (Biolegend). Cells were then fixed and permeabilised using the Cytofix/Cytoperm kit (BD). Intracellular staining was performed for 30 min at 4 °C with the following antibodies diluted in Perm/wash buffer (BD): anti-CD3 APC (BD), anti-IL-9 PE (Biolegend) and anti-IFN-γ PECy7 (Biolegend). Lung cells were also stained for mast cell detection with anti-CD45 PercP and anti-CD117 APC (Biolegend). Samples were acquired using a FACS Canto II (BD) and analysed using FlowJo software (version 9.0.2, Tree Star).
4
Multiparameter Flow Cytometry Profiling
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After cell culture, whole blood samples (10x105 cells/mL) were incubated with the following panel: antibodies from BioLegend, San Diego, CA, USA: anti-CD45-PerCP (Clone: HI30), anti-CD3-AF647 (Clone: UCHT1), anti-CD14-PECy7 (Clone: M5E2), anti-CD4-APC/Cy7 (Clone: OKT4), anti-CD8-PE/Dazzle594 or BV510 (Clone: SK1). After 15 min in the dark, blood was washed once with PBS (1 mL) by centrifugation at 900×g for 5 min at room temperature (RT); then, Fixation buffer was added (100 μL, Cat: 420801, BioLegend, San Diego, CA, USA), and the samples were incubated for 20 min. Then, samples were washed twice with 1 mL of Intracellular Staining Perm Wash buffer (Cat: 421002, BioLegend, San Diego, CA, USA); after the second wash, they were mixed with monoclonal antibodies against cytokines from BioLegend, San Diego, CA, USA: anti-TNFα-BV421 (Clone: MAb11), anti-IL-6-PE (MQ2-13A5), anti-IL-1β-FITC (Clone:JK1B-1), anti-IFNγ-BV421 (Clone:4S. B3), anti-IL-8a-PE (Clone: E8N1). For the exclusion of dead cells, a Zombie Aqua fixable viability kit (BioLegend, San Diego, CA, USA) was added and incubated for 30 min at RT. Last, the mixture was washed once with PBS. At least 30,000 events were acquired in a FACSAria IIu (BD, San Jose CA) flow cytometer. Analysis was performed using Infinicyt™ Software 1.8.
5
In Vitro and In Vivo Phagocytosis Assays
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The in vitro phagocytosis assays were performed by coculturing target GFP-positive cells and macrophages at a ratio of 120,000 target cells to 60,000 macrophages for 1–2 h in ultralow-attachment 96-well plates (Cat No. 3473, Corning, USA) in serum-free RPMI-1640 medium in a humidified 5% CO2 incubator at 37 °C. After co-culture, reactions were centrifuged at 960 × g for 2 min at 4 °C, and then stained with an APC-labelled anti-CD45 antibody (Cat No. 982314, BioLegend, USA) to identify human macrophages. Cells were analyzed by flow cytometry on a BD FACSCanto ΙΙ instrument. Phagocytosis was quantified as the number of CD45+ and GFP+ macrophages as a percentage of the total number of CD45+ macrophages.
For the in vivo phagocytosis assay, cells were harvested by peritoneal lavage as described above. Cells were stained with anti-CD45-PerCP (Cat No. 103132, BioLegend, USA), anti-CD11b-APC (Cat No. 101212, BioLegend, USA), and anti-F4/80-PE (Cat No. 123110, BioLegend, USA) antibodies. Data were collected with a FACSCalibur flow cytometer (BD Biosciences). Phagocytosis was quantified as the percentage of CD11b+F4/80+ TAMs that were also GFP-positive. The gating strategy is shown in Fig.S2 .
For the in vivo phagocytosis assay, cells were harvested by peritoneal lavage as described above. Cells were stained with anti-CD45-PerCP (Cat No. 103132, BioLegend, USA), anti-CD11b-APC (Cat No. 101212, BioLegend, USA), and anti-F4/80-PE (Cat No. 123110, BioLegend, USA) antibodies. Data were collected with a FACSCalibur flow cytometer (BD Biosciences). Phagocytosis was quantified as the percentage of CD11b+F4/80+ TAMs that were also GFP-positive. The gating strategy is shown in Fig.
6
Comprehensive Leukocyte Profiling by Flow Cytometry
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Fresh whole-blood leukocytes were analyzed by flow cytometry to identify and quantify the major leukocyte populations. Briefly, 200 μl EDTA-stabilized venous blood was stained with a panel of fluorochrome-conjugated antibodies (anti-CD45-PerCP [Clone: HI30; BioLegend], anti-CD3-APC/Cy7 [Clone: OKT3; BioLegend], anti-CD4-BUV395 [Clone: SK3; BD Biosciences], anti-CD8-BUV737 [Clone: SK1; BD Biosciences], anti-CD19-PE [Clone: HIB19; BioLegend], anti-CD14-Alexa Fluor 488 [Clone: HCD14; BioLegend], anti-CD16-PE/Cy7 [Clone: 3G8; BioLegend], anti-CD15-BV605 [Clone: W6D3; BioLegend], and anti-CD56-APC [Clone: HCD56; BioLegend]) for 15 min at room temperature in BD Truecount tubes. Red blood cells were lysed (BD FACS Lysing solution), and leukocytes were analyzed immediately on a BD Fortessa flow cytometer with BD FACSDiva software. Absolute cell numbers were calculated according to the manufacturer’s instructions (Truecount Tubes, BD Biosciences).
For the characterization of both rare and abundant leukocyte populations in the blood, cryopreserved PBMCs (4–8 × 105 cells per individual) from the PD-L1-deficient siblings, their heterozygous mother, a healthy adult, and age-matched controls, and the previously described PD-1-deficient child (Ogishi et al., 2021 (link)) were analyzed by spectral flow cytometry as previously described (Ogishi et al., 2023 (link)).
For the characterization of both rare and abundant leukocyte populations in the blood, cryopreserved PBMCs (4–8 × 105 cells per individual) from the PD-L1-deficient siblings, their heterozygous mother, a healthy adult, and age-matched controls, and the previously described PD-1-deficient child (Ogishi et al., 2021 (link)) were analyzed by spectral flow cytometry as previously described (Ogishi et al., 2023 (link)).
7
Isolation and Culture of Mouse BMSCs
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C57BL/6J mice were anesthetized with pentobarbital and then sacrificed. The tibias and femurs of the mice were separated, and the ends of the tibias and femurs were cut. The bone marrow was flushed out with a 10 ml syringe with a 25-gauge needle filled with 10 ml of 1× PBS. The cell suspensions were centrifuged at 700 × g for 5 minutes at 4°C, and the cells were resuspended in 500 μl of 1× PBS. Then, the cell suspensions were incubated with anti-Sca-1-PE (BioLegend, USA), anti-CD29-FITC (BioLegend, USA), anti-CD45-PerCP (BioLegend, USA), and anti-CD11b-PerCP (BioLegend, USA) antibodies for 30 minutes at 4°C. The mouse BMSCs (Sca-1+CD29+CD45-CD11b-) were sorted by fluorescence-activated cell sorting (FACS) with a FACSAria (BD Biosciences, USA) and cultured with α-MEM culture medium (Cellmax, China) with 10% fetal bovine serum (FBS) and 1% streptomycin (Gibco, USA) and penicillin (Gibco, USA) at 37°C in a humidified atmosphere of 5% CO2.
8
Isolation and Expansion of Human CD34+ HSPCs
9
Isolation and Analysis of Myeloid-Derived Suppressor Cells
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Freshly removed spleens were mechanically disaggregated and tumor tissues from mouse xenograft model were mechanically minced into small pieces and digested in a 1‐mL mixture of 300 U·mL−1 collagenase IV (Roche, Basle, Switzerland), 300 U·mL−1 collagenase type I (Sigma, St. Louis, MO, USA) and 0.4 mg·mL−1 DNase I in RPMI 1640 medium at 37 °C for 1 h. After the incubation, the cell suspensions were filtered through a 70‐μm mesh and then washed with complete RPMI medium prior to immunostaining. Peripheral blood mononuclear cells (PBMC) from mice were first isolated by mouse PBMC separation solution kit (Hao Yang Biological Manufacturers, Tianjin, China) for detecting myeloid‐derived immunosuppressive cells (MDSC). The single‐cell suspensions isolated from the spleens and tumor were first incubated with Fc‐blocker anti‐CD16/32 antibody (dilution 1 : 20; Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min, followed by staining after pre‐incubation with anti‐CD45‐PerCP (BioLegend, San Diego, CA, USA), anti‐CD11b‐APC (1 μL per test) and anti‐GR‐1‐PE (0.3 μL per test) for 30 min at 4 °C in the dark. Cells were then washed with buffer to remove the excess stains and analyzed by FACS (Becton Dickinson, San Diego, CA, USA).
10
Multicolor Flow Cytometry of Tumor B Cells
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PBMC and tumor single cell suspensions were stained with LIVE/DEAD Fixable Aqua (Invitrogen) according to the manufacturer’s instructions and then incubated with Fc Blocking Reagent (Miltenyi Biotec) for 10 min at room temperature followed by staining with anti-CD45 PerCP (BioLegend), anti-CD19 FITC (BD), anti-CD27 BV421 (BD) and anti IgD APC H7 (BD) (BioLegend) for 30 min at 4 °C. The samples were then washed in Phosphate-Buffered Saline (PBS) 2% FBS (FACS Buffer), resuspended in 200 µl of FACS Buffer and analyzed by multicolor flow cytometry using BD FACSCanto II (BD Biosciences). Tumors without detectable B cells (less than 10 per sample) were excluded from analyses. For Ig sequence analysis, CD27+IgD- B cells (CD45 + CD19+) were sorted on 96-well plates at 1, 5, and 10 cells/well directly into 10 µl of lysis buffer (Single cell lysis kit, Ambion-Thermo Fisher Scientific), using BD FACSAria II Cell Sorter (BD Biosciences) with a 70 µm nozzle.
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