MC38 cells (∼80% confluent) were split into flat 96-well tissue culture plates (Thermos, catalog no. 167008) at a density of 8 × 104 cells in 200 µl complete culture media (DMEM + 10%FBS + 1%Pen/Strep) per well, and cultured overnight. After gently aspirating culture media, 4 × 105 erythrocyte-depleted splenocytes prepared from FcgRα−/−, or hFCGRTg B6 mice and, resuspended in 100 µl complete culture media, 100 µl of culture media containing 1 µg ml−1 of control IgG (Jackson ImmunoResearch Laboratories, catalog no. 009-000-003), αDR5:hIgG1, αDR5:hIgG2, αDR5:hIgG3, αDR5:hIgG4, αDR5:hIgG V11(H1), or αDR5:hIgG V11(H3) were added with or without 1 µg ml−1 of 2B6. Four hour later, cells were all harvested and stained with anti-mouse CD45.2 (BD, catalog no.560691), followed by Annexin V/PI using Annexin V FITC Apoptosis Detection Kit I (BD Biosciences, catalog no.556547) or intracellular active caspase-3 (clone C92-605; BD Biosciences) staining according to manufacturer’s instructions. Samples were analyzed with a BD FACSCaliburTM or LSRFortessaTM X-20 flow cytometer. MC38 cells were gated based forward, side scatters and the lack of CD45.2-expression, and analyzed for percentage of Annexin V+PI− or Actived caspase-3+ apoptosis cells (Supplementary Fig. 8c , d ).
Anti mouse cd45
Manufactured by BD
Sourced in United States
Anti-mouse CD45 is a laboratory reagent used for the detection and analysis of CD45, a transmembrane glycoprotein expressed on the surface of most hematopoietic cells in mice. It is commonly used in flow cytometry and other immunological applications to identify and characterize different cell populations.
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Lab products found in correlation
Lsr 2, by BD (3 mentions)
Anti mouse cd4, by BD (3 mentions)
Anti mouse cd206, by BD (3 mentions)
Facscalibur, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions)
Anti mouse il 2, by BD (2 mentions)
Rabbit anti mouse hif1α antibody, by Novus Biologicals (2 mentions)
Mouse anti mouse tfam, by Santa Cruz Biotechnology (2 mentions)
Anti mouse tcf7 tcf1 antibody, by R&D Systems (2 mentions)
Facscanto 2, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Anti mouse ifn γ, by BioLegend (2 mentions)
Anti mouse cd8, by BioLegend (2 mentions)
Anti mouse f4 80, by BioLegend (2 mentions)
Anti human cd33, by BD (2 mentions)
7 aad, by BD (2 mentions)
Anti cd3, by BD (2 mentions)
Anti cd8, by BD (2 mentions)
32 protocols using anti mouse cd45
1
Apoptosis Assay for Antibody-Mediated DR5 Activation
2
Quantification of Effector Memory CD8+ T Cells
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Surface stained Ova-specific effector memory CD8+ T cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences), followed by staining with rabbit anti-mouse HIF1α antibody (Novus Biologicals#nb100-449) or mouse anti-mouse TFAM (Santa Cruz Biotechnology#sc-166965) at 4°C for 1 hour. This was followed by staining with Alexa Fluor conjugated secondary antibodies (Molecular Probes) at 4°C for 1 hour. In case of intracellular staining of IL-2 and IFN-γ, splenocytes harvested from CD45.1+ mice were pre-treated with FcRγII/III (Fc blocker) and IgG2b anti-mouse CD16/CD32 antibodies, then stained with anti-mouse CD8 (Biolegend#100725), anti-mouse CD45.2 (BD PharMingen#561096) and anti-mouse Ova_tetramer before intracellular staining. Surface stained splenocytes were then fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences#554714), followed by staining with anti-mouse IL-2 (BD PharMingen#554428) or anti-mouse IFN-γ (Biolegend# 505806). In case of intranuclear staining for TCF7, surface stained cells were fixed and permeabilized using Foxp3/Transcription factor staining buffer set (eBioscience#00-5523-00), followed by staining with anti-mouse TCF7/TCF1 antibody (R&D systems#FAB8224R). Stained cells were analyzed on BD FACSCantoII or BD LSRII.
3
Quantification of Effector Memory CD8+ T Cells
4
Immune Reconstitution in Humanized Mouse Model
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Human immune cell reconstitution in hu-mice was analyzed by flow cytometry (FCM) using various combinations of the following monoclonal antibodies: anti-human CD45, CD3, CD4, CD8, CD45RA, CD45RO, CD69, CCR7, CD31 (all purchased from Biolegend, San Diego, CA, USA); and anti-mouse CD45 (BD Pharmingen) and Ter119 (Biolegend, San Diego, CA, USA). Peripheral blood was collected from tail vein into heparinized tubes, and mononuclear cells were purified by density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). FCM analysis was performed on a FACS Fortessa (BD Biosciences). Dead cells were excluded from the analysis by gating out lower forward scatter and high propidium iodide–retaining cells. Data analysis was performed using FlowJo 10.3 software.
5
Quantifying Immune Cell Populations
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The number of neutrophils, B cells, and CD4 + T cells was analyzed by flow cytometry. Briefly, peripheral blood mononuclear cells (PBMCs) from mice in different groups were stained with anti-mouse CD45 (BD Biosciences, 561,037, USA), anti-mouse CD4 (BD Biosciences, 553,651, USA), anti-mouse B220 (ebioscience, 69–0452-82, USA), anti-mouse CD11b (BD Biosciences, 550,993, USA), anti-mouse Ly6G(BD Biosciences, 560,599, USA), anti-mouse Ly6C (ebioscience, 25–5932-80, USA) for 30 min. After washing with PBS, the cells were analyzed with BD FACSCanto II flow cytometer.
6
Flow Cytometry Protocol for Cell Characterization
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The detailed flow cytometry procedure was performed as previously described [31 (link)]. The following antibodies were used: anti-mouse IFNγ (BD Biosciences), anti-mouse CD45 (BD Biosciences), anti-mouse Gr-1 (TONBO Biosciences), anti-mouse Ly6G (TONBO Biosciences), anti-mouse ly6C (BD Biosciences), anti-mouse CD11b (TONBO Biosciences), anti-mouse F4/80 (BioLegend), anti-human CD11b (TONBO Biosciences), anti-human CD33 (BD Biosciences), and anti-human HLA-DR (BD Biosciences). Detailed information on the antibodies is provided in Additional file 2 : Table S2. Briefly, the cells were digested and suspended as single cells, washed with PBS, and then resuspended in cell stabilizing buffer (BioLegend cat. No. 420201); the supernatant was discarded, and the sample was centrifuged at 350 g for 5 min. Then, 5 μl of Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301) and the antibodies were added and cultured for 15-20 min at room temperature. Finally, the cells were assessed by flow cytometry. For intracellular staining of IFNγ, fixation buffer (BioLegend Cat. No. 420801) was added and incubated for 20 min at room temperature. Then, the cells were washed with Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002), resuspended and centrifuged. IFNγ antibody was added, and the cells were cultured for 20 min and finally assessed by flow cytometry.
7
Multiparameter Flow Cytometry Analysis
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Fluorochrome-conjugated isotype controls, anti-CD123, anti-CD3, anti-CD4, anti-CD8, anti-CD33, anti-CD45, anti-CD45RA, anti-CD62L, anti-CCR7, anti-PD-1, anti-TIM3, anti-LAG3, anti-Annexin-V, 7-AAD, DAPI, and anti-mouse CD45 were purchased from BD Biosciences or BioLegend (San Diego, CA). Analysis was performed on at least 20,000 cells per sample using a LSR II flow cytometer (BD Biosciences) and a Gallios Flow Cytometer, and analyzed using Kaluza Analysis Software (Beckman Coulter, Indianapolis, IN).
8
Murine Tumor Immune Profiling
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For murine samples, mice with tumors were killed according to the institutional ethical guidelines and femurs, tibias, and tumors were collected. Signal cell of suspension of tumor was obtained by using the Tumor Dissociation Kit (Miltenyi Biotech, Bergish Gladbach, Germany, 130-096-730) according to the manufacturer’s instructions. Antibodies (BD Pharmingen, San Diego, CA, USA) used are listed: anti-mouse-CD3e (BD Pharmingen, 566494), anti-mouse-CD45 (BD Pharmingen, 553079), anti-mouse-CD8e (BD Pharmingen, 553035), anti-mouse-CD11b (BD Pharmingen, 550993), anti-mouse-Ly-6G/Ly-6C (BD Pharmingen, 553129), Anti-mouse-F4/80 (BD Pharmingen, 565410). For cell apoptosis assays, apoptosis cells were measured by a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Data were acquired using FACS Calibur (BD Biosciences), and analyzed using Cell Quest Pro software (BD Biosciences)46 (link).
9
Eosinophil Quantification in BALF Samples
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Cells were collected from bronchoalveolar lavage fluid (BALF) and pre-incubated with Fc-blocking anti-mouse CD16/32 antibody (BD Pharmingen, #553,141) before the staining process. Subsequently, dead cells were excluded by DAPI staining (BD Pharmingen, #564,907). The cells then underwent surface staining with anti-mouse CD45 (BD Pharmingen, #561,037), anti-mouse CD11b (BD Pharmingen, #561,688), anti-mouse GR-1 (BD Pharmingen, #561,103), anti-mouse CD-11c (BD Pharmingen, #561,022), and anti-mouse MHCII (Biolegend, #107,613) for 30 min at 4 °C. All samples were analyzed using Cytek Dxp Athena flow cytometer, and the data were processed using FlowJo software (version 10). The method for eosinophil count was conducted following established protocols from previous study [23 (link)], and is illustrated in Fig. S1 . Initially, live leukocytes were selected based on the expression of CD45 and exclusion of DAPI, effectively removing debris, erythrocytes, and dead cells. Subsequently, lymphocytes were differentiated through the analysis of the SSC-A/CD11b plot, while neutrophils were identified using the SSC-A/GR1 plot among the diverse cell populations. Ultimately, eosinophils were isolated from the remaining cells using the MHC-II/CD11c plot.
10
Tracking Engraftment of HSPC Transplantation
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Mouse peripheral blood (mPB) samples were collected from the retro-orbital plexus or tail vein 8 weeks after CD34+ HSPC transplantation. At 15 weeks after transplantation, the mice were euthanized by CO2, and bone marrow (mBM) and mPB were harvested. mBM was flushed from the femur in RPMI with 10% FBS. Red blood cells were lysed from the PB and BM samples using RBC lysis buffer (Biolegend, San Diego, CA). Single cell suspensions of mPB and mBM were labeled with anti-mouse CD45 (#552848, BD Biosciences), anti-human CD45 (#563879, BD Biosciences), anti-human CD3 (#555341, BD Biosciences), anti-human CD14 (#557831, BD Biosciences), anti-human CD20 (#555623, BD Biosciences), and anti-human CD33 (#564588, BD Biosciences) antibodies and run on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA), with analysis by FlowJo V.10 software (BD Biosciences, Franklin Lakes, NJ, USA). To exclude dead cells, 7-AAD (#559925, BD Biosciences) was used.
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