The following anti-mouse antibodies were used for flow cytometric analysis: anti-CD4 (clone RM4-5, Biolegend); anti-CD8a (clone 53-6.7, Biolegend); anti-CD45.1 (clone A20, Biolegend); anti-CD45.2 (clone 104, Biolegend); anti-CD25 (clone PC61, Biolegend); anti-CD45RB (clone 16A, BD Biosciences); anti-B220 (clone Ra3-6B2, BD Biosciences); anti-CD11c (clone L3, BD Biosciences); anti-Thy1.1 (clone OX-7, Biolegend); anti-Thy1.2 (clone 53-2.1, Biolegend); anti-CCR7 (clone 4B12, Biolegend). Antibodies for total eIF2α and p-Ser51 eIF2α were from cell Signaling (clones D7D3 and D9G8 rabbit XP mAbs). OVA 257–264 and OVA 323–339 peptides were purchased from American Peptide Company. Live/Dead Fixable Violet was purchased from Invitrogen.
Anti cd45.2 clone 104
Manufactured by BioLegend
Sourced in United States
Anti-CD45.2 (clone 104) is a mouse monoclonal antibody that specifically binds to the CD45.2 isoform of the CD45 cell surface glycoprotein. CD45 is a receptor-like protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b clone m1 70, by BioLegend (5 mentions)
Anti cd45.1 clone a20, by BioLegend (4 mentions)
Anti cd3e clone 145 2c11, by BioLegend (4 mentions)
Anti cd25 clone pc61, by BioLegend (3 mentions)
Anti cd4 clone rm4 5, by BioLegend (3 mentions)
Anti cd4 clone gk1.5, by BD (3 mentions)
Anti cd8 clone 53 6, by BD (3 mentions)
Calibrite apc beads, by BD (3 mentions)
Anti cd44 clone im7, by BioLegend (3 mentions)
Anti cd49b clone dx5, by BioLegend (2 mentions)
Anti cd127 clone a7r34, by BioLegend (2 mentions)
Anti ifn γ clone xmg1, by BioLegend (2 mentions)
Anti cxcr3 clone cxcr3 173, by BioLegend (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Facscalibur, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti b220 clone ra3 6b2, by BD (1 mentions)
Anti cd45rb clone 16a, by BD (1 mentions)
Anti cd8a clone 53 6.7, by BioLegend (1 mentions)
Live dead fixable violet, by Thermo Fisher Scientific (1 mentions)
13 protocols using anti cd45.2 clone 104
1
Flow Cytometry Analysis of Mouse Immune Cells
2
Mast Cell Surface Marker Analysis
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The surface expressions of FcεRI, CD117 (c-kit), ST2, β-Integrin and CD49b were analyzed by flow cytometry. Briefly, 1 × 106 mast cells were washed with PBS and incubated with an anti-CD16/CD32 mAb (clone 32, Biolegend, London, UK) for Fc receptor blocking. The following primary, fluorochrome-coupled antibodies were used for analysis: anti-CD45.2 (clone 104, Biolegend), anti-FcεRI (clone MAR-1, Biolegend), anti-CD117 (clone 2B8, Biolegend), anti-ST2 (clone DIH4, Biolegend), anti-integrin-ß7 (clone FIB27, Biolegend) and anti-CD49b (clone DX5, Biolegend) and Live/Dead fixable aqua dead cell stain (Invitrogen Thermo Fischer, Munich, Germany). Cells were determined fully matured if >90% were FcεRI+CD117+ and only then further studies were conducted.
3
Quantification of Spinal Cord Immune Cells
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Single-cell suspensions from spinal cords were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient. Staining of αβTCR/CD4+ T cells, αβTCR/CD8+ T cells and CD45/CD11b cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution: Anti-CD3e (clone 145-2C11), BioLegend; anti-CD4 (clone GK 1.5), BD; anti-CD8 (clone 53-6.7), BD; anti-CD8 (clone 53–6.7), BD; anti-CD11b (clone M1/70), BioLegend; anti-CD45.2 (clone 104), BioLegend. The addition of Calibrite APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a FACSCalibur operated by Cell Quest software (Becton Dickinson).
4
Flow Cytometry Antibody Panel for Immune Cell Profiling
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Antibodies to following antigens were used for flow cytometry: anti-CD4 (clone RM4-5, Biolegend #100536), anti-CD5 (clone 53-7-3, Biolegend #100627, #100625), anti-CD8α (clone 53-6.7, Biolegend #100738), anti-CD24 (clone M1/69, Biolegend #101806), anti-CD25 (clone PC61, Biolegend #102016 and #102036), anti-CD44 (clone IM7, Biolegend #103049), anti-CD45.1 (clone A20, Biolegend #110723), anti-CD45.2 (clone 104, Biolegend #109808), anti-CD49d (clone R1-2, Biolegend #103618), anti-CD127 (clone A7R34, Biolegend #135012), anti-TCRβ (clone H57-597, Biolegend #109218), anti-KLRG1 (clone 2F1, Biolegend #138421), anti-Ly6C (clone HK1.4, Biolegend #128006), biotin-conjugated anti-BST2 (clone 927, Biolegend #127006), anti-biotin (clone 1D4-C5, Biolegend #409004). Viability was stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen #L34975), Zombie Green™ Fixable Viability Kit (Biolegend, #423111) or Hoechst 33258 (Invitrogen, #H3569). For the analysis of Jurkat cell lines, anti-CD8 (clone MEM-31, Exbio #1P-207-T025), anti-CD69 (clone FN50, Exbio #T7-552-T100) were used. Antibodies were conjugated with various fluorophores and used according to the manufacturer’s instructions.
5
Analyzing Antigen-specific CD8 T Cells
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Antigen-specific CD8 T cells were identified by labeling with Db/NP366 or Db/PA224in house prepared tetramers for 1 h at 4°C (22 (link)). Tetramer staining was followed by surface staining with appropriate antibody cocktails for 20 min at 4°C. Surface markers were stained using following antibodies: anti-CD8 (clone 53-6.7, BioLegend), anti-CD90.2 (clone 30-H12, BioLegend), anti-CD45.2 (clone 104, BioLegend), anti-CD103 (clone 2E7, BioLegend), anti-CD69 (clone H.12F3, BioLegend), anti-KLRG-1 (clone 2F1, eBioscience, San Diego, CA, USA), anti-CD127 (clone A7R34, BioLegend), anti-CX3CR1 (clone SA011F11, BioLegend), anti-CXCR3 (clone CXCR3-173, BioLegend), and anti-CD49a (clone Ha31/8, BD Pharmingen). Intracellular cytokine staining was performed using anti-IFNγ (clone XMG1.2, BioLegend), anti-TNF (clone MP6-XT22, BioLegend), and anti-IL2 (clone JES6-5H4, BioLegend) antibodies. Proliferation of CD8 T cells was assessed by intracellular staining with anti-Ki67 (clone MOPC-21, BD Pharmingen). Flow cytometry data were acquired using LSRFortessa (Becton Dickinson, Rutherford, NY, USA) and analyzed using the FlowJo software (Tree Star Inc., Ashland, OR, USA).
6
Multicolor Flow Cytometry Analysis
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The following monoclonal antibodies were used: FcR‐blocking antibody (anti‐mouse CD16/CD32, clone 2.4G2; BD Pharmingen), FITC, PE, PerCP, or PerCP/Cy5.5‐conjugated anti‐CD335 (clone 29A1.4; BioLegend), anti‐CD49b (clone HMα2; BioLegend), anti‐CD11b (clone M1/70; BioLegend), anti‐CD4 (clone RM4‐5; eBioscience), anti‐CD8a (clone 53‐6.7; eBioscience), anti‐CD45.2 (clone 104; BioLegend), anti‐IFN‐γ (clone XMG1.2; BioLegend), Rat IgG2a,k (BD Pharmingen), Rat IgG2b, k control (BD Pharmingen), Rat IgG1k isotype‐matched control Abs (BioLegend), and PE/Cy7‐conjugated Armenian hamster IgG (BioLegend) Abs.
Cells were labeled at 4°C with specific antibodies. For intracellular cytokine analysis, cells were resuspended in RPMI‐1640 medium supplemented with 20% FBS, and phorbol 12‐myristate 13‐acetate (50 ng/mL), ionomycin (500 ng/mL), and brefeldin A (10 μg/mL; all from Sigma) were added. After incubation for 4 h, the cells were washed, stained for surface molecule markers, and fixed and permeabilized using IntraPrep reagent (Beckman Coulter), followed by intracellular staining of IFN‐γ. The stained cells were analyzed using a FACSCalibur (BD Biosciences).
Cells were labeled at 4°C with specific antibodies. For intracellular cytokine analysis, cells were resuspended in RPMI‐1640 medium supplemented with 20% FBS, and phorbol 12‐myristate 13‐acetate (50 ng/mL), ionomycin (500 ng/mL), and brefeldin A (10 μg/mL; all from Sigma) were added. After incubation for 4 h, the cells were washed, stained for surface molecule markers, and fixed and permeabilized using IntraPrep reagent (Beckman Coulter), followed by intracellular staining of IFN‐γ. The stained cells were analyzed using a FACSCalibur (BD Biosciences).
7
Comprehensive Immune Cell Profiling by Flow Cytometry
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Flow cytometry was performed as previously described Fang et al. (2010 (link)). The following antibodies were used: anti-CD3e (clone 145-2C11, Biolegend), anti-CD11b (clone M1/70, Biolegend), anti-CD19 (clone 1D3, BD Biosciences), anti-CD27 (clone LG.3A10, Biolegend), anti-CD29 (clone HMβ1-1, Biolegend San Diego, CA), anti-CD44 (clone IM7, Biolegend), anti-CD45.1 (clone A20, Biolegend), anti-CD45.2 (clone 104, Biolegend), anti-CD49a (clone HMα1, Biolegend), anti-CD49b (clone DX5, Biolegend), anti-CD105 (clone MJ7/18, Biolegend), anti-CD106 (clone 429, Biolegend), anti-BrDU (clone PRB-1, eBioscience), anti-CXCR3 (clone CXCR3-173, Biolegend), anti-Eomes (clone Dan11mag, eBioscience), anti-KLRG1 (clone 2F1/KLRG1, Biolegend), anti-Ly-6A/E (Sca-1) (clone E13-161.7, Biolegend), anti-Ly49A (clone A1/Ly49A, Biolegend), anti-Ly49C/I (clone 5E6, Biolegend), anti-Ly49D (clone 4E5, Biolegend), anti-Ly49G2 (clone LGL-1, eBioscience), anti-Ly49H (clone 3D10, Biolegend), anti-NK1.1 (clone PK136, Biolegend), NKG2A/C/E (clone 20d5, Biolegend), anti-Tbet (clone 4B10, Biolegend), and anti-TRAIL (clone N2B2, Biolegend). At least 500 000 cells were analyzed by flow cytometry at the Fox Chase Cell Sorting Facility using an LSR II system (BD Biosciences).
8
Murine Splenocyte Isolation and Flow Cytometry
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Splenocyte suspensions were prepared by passing the spleens directly through a 70 μM filter or by first dissecting them into small pieces and digesting them in RPMI containing 1 mg/ml Liberase (Roche) and 0.2 mg/ml DNase I (Roche) for 30 min at 37°C, prior to filtration. Red blood cells were removed using lysis buffer (Biolegend), and then cells were washed by centrifugation in PBS containing 1% foetal calf serum.
Single‐cell suspensions were incubated for 30 min at 4°C with Fc Block (anti CD16/CD32 clone 93, Biolegend) and H‐2Db tetramer (N396‐404) from the NIH Core Tetramer Facility. Cells were next stained with cell surface markers for 30 min at 4°C. The mAbs used were anti‐CD45.1 (clone A20, Tonbo Biosciences), anti‐CD45.2 (clone 104, Biolegend), anti‐CD8a (clone 53‐6.7, BD Pharmingen), anti‐CD3 clone (17A2, Biolegend), anti‐CD44 (clone IM7, BD Horizon) and anti‐PD1 (clone J43, BD Pharmingen). Samples were acquired on an LSRFortessaTM (BD Biosciences). The data were analysed using FlowJo software.
Single‐cell suspensions were incubated for 30 min at 4°C with Fc Block (anti CD16/CD32 clone 93, Biolegend) and H‐2Db tetramer (N396‐404) from the NIH Core Tetramer Facility. Cells were next stained with cell surface markers for 30 min at 4°C. The mAbs used were anti‐CD45.1 (clone A20, Tonbo Biosciences), anti‐CD45.2 (clone 104, Biolegend), anti‐CD8a (clone 53‐6.7, BD Pharmingen), anti‐CD3 clone (17A2, Biolegend), anti‐CD44 (clone IM7, BD Horizon) and anti‐PD1 (clone J43, BD Pharmingen). Samples were acquired on an LSRFortessaTM (BD Biosciences). The data were analysed using FlowJo software.
9
Multicolor Flow Cytometry of Pancreatic ROS
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Flow cytometry slides were stained with the following antibodies: anti-CD45.2 (clone 104,1:200, Biolegend, San Diego, CA, USA), anti-CD11b (clone M1/701:200, Biolegend), anti-Ly6G (clone 1A8,1:200, Biolegend), anti-myeloperoxidase (clone EPR20257,1:500, Abcam), and MPO coupling Alexa Fluor488 (ab150077,1:1000, Abcam). The ROS content in the pancreatic tissue samples was detected using a DHE fluorescent probe (Sigma, St. Louis, MO, USA, D70N8, 1:2000). The cells were stained with DHE solution and incubated at 37 °C for 30 min. Cells were washed twice with PBS, then stained again with the above flow cytometry antibodies, and incubated at 4 °C for 30 min. Finally, the cells were collected using a Beckman DxFlex B5-R3-V3(Indianapolis, IN, United States) and analyzed using CytExpert2.4.0.28 for DxFlex.
10
Immunophenotyping of Heart and Spleen
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Heart grafts and spleens were homogenized and were stained with the following antibodies: anti-CD8a (53–6.7), anti-CD44 (IM7), anti-CD25 (PC61), anti-CD11c (N418), anti-B220 (RA3-6B2) (BD Biosciences), anti-F4/80 (BM8), anti-Foxp3 (FJK-16S), anti-CD4 (GK1.5) (Thermo Fisher Scientific), anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD45.1 (A20), and anti-CD45.2 (clone:104) (BioLegend) (S1 Table ). 7-Aminoactinomycin D (7-AAD; BD Biosciences) was added to stain dead cells. Flow cytometry was performed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
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