To visualize immunological synapses, 200 μL of U87MG cells (50,000 cells/mL) were seeded onto wells of a 24-well plate and incubated at 37 oC for 12 h to allow cells to attach. 200 μL neutrophils (500,000 cells/mL) were then added onto the target U87MG cells and incubated for 6 h before fixation with 4% paraformaldehyde (in PBS). Cytoskeleton staining was then performed using an F-actin Visualization Biochem Kit (Cytoskeleton Inc.). To quantify the immunological synapses, a total of 1 × 106 CAR hPSC-neutrophils or CAR hPSC-neutrophils@R-SiO2-TPZ labeled with anti-CD45-APC (BD Biosciences) were incubated with 2 × 105 targeted cells stained with Calcein-AM fluorescent dye (Invitrogen) for various time points at 37oC and humidified 5% CO2 atmosphere. After incubation, the cells were fixed and analyzed in an Accuri C6 plus cytometer (Beckton Dickinson) after washing with BSA-containing PBS−/− solution, and cells of double-positive events APC + /Calcein+ were analyzed to quantify immunological synapse formation in FlowJo software.
Anti cd45 apc
Manufactured by BD
Sourced in United States
Anti-CD45-APC is a monoclonal antibody conjugated with allophycocyanin (APC) that binds to the CD45 surface antigen expressed on various hematopoietic cells. It is a tool used in flow cytometry applications for the identification and analysis of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 fitc, by BD (5 mentions)
Anti cd31 apc, by BD (4 mentions)
Facsaria 2, by BD (4 mentions)
Accuri c6, by BD (4 mentions)
Anti cd34 fitc, by BD (3 mentions)
Anti cd73 pe, by BD (3 mentions)
Anti cd29 pe, by BD (3 mentions)
Anti cd3 percp cy5, by BD (3 mentions)
Anti cd14 fitc, by BD (3 mentions)
Anti ly6c fitc, by BD (2 mentions)
Anti cd90 apc, by BD (2 mentions)
Anti ve cadherin, by Abcam (2 mentions)
Anti vinculin, by Santa Cruz Biotechnology (2 mentions)
Anti cd45ro, by BD (2 mentions)
Anti fak, by Abcam (2 mentions)
Texas red phalloidin, by Thermo Fisher Scientific (2 mentions)
Efluor 670, by BD (2 mentions)
Anti pd 1, by BD (2 mentions)
Anti lag3, by BD (2 mentions)
Anti ccr7, by BD (2 mentions)
45 protocols using anti cd45 apc
1
Quantifying Immunological Synapse Formation
2
Lung Immune Cell Isolation and Flow
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The left lobes of the lung tissue were pooled by each group and slightly chopped using a pair of scissors in cold RPMI 1640 containing 1 μg/ml of dipase II (Sigma Aldrich, St. Louis, MO, USA). Samples were incubated at 37°C, 1,100 RPM shaking chamber. After 1 h, samples were placed on 40-μm cell strainer and homogenized using syringe rubber to obtain the single cells. RBCs were lysed using RBC lysis buffer, and flow cytometry assay was performed as previously described (31 (link)). In briefly, cells were stained with anti-CD45-APC (BD Biosciences, San Jose, CA, USA), anti-CD11c-PE (BD Biosciences), and anti-F4/80-FITC (Invitrogen, Carlsbad, CA, USA). Analysis was performed by using MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany).
3
Expanded hSDSCs and pSDSCs Characterization
4
Antibody Panel for Immune Cell Profiling
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Antibodies were purchased from Santa Cruz Biotechnology (USA), BD Biosciences (USA) and Imgenex (Bhubaneswar, Odisha, India). Anti-MBP antibody (Cat No. 808) was from Santa Cruz. Anti-CD3-FITC (Cat No. 555274), anti-CD8-APC-Cy7 (Cat No. 557654), anti-CD19-APC (Cat No. 550992), anti-CD25-PECy7 (Cat No. 552880) and anti-CD45-APC (Cat No. 559864) were from BD Biosciences. Anti-CD4-PE (Cat No. 5922D) was purchased from Imgenex.
5
Multiparameter Flow Cytometry Analysis
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Three to ten million cells were transferred to a clean 15 mL conical tube, and the volume was adjusted to 1 mL using 0.5% BSA/PBS solution. Next, five tubes each containing 100,000 cells in 100 µL 0.5% BSA/PBS solution were prepared for individual staining/negative control. The following antibodies/dyes were used (according to the manufacturers' instructions): anti-EPCAM-PE (cat. no. 347198, Becton Dickinson (BD), Franklin Lakes, NJ, USA), anti-CD31-Alexa488 (cat. no. 558068, BD), anti-CD45-APC (cat. no. 560973, BD) antibodies, and Live-Dead-NearIR dye (cat. no. L34961, Life Technologies, Thermo Fisher). The antibody-sample mix was incubated at room temperature (RT) for 30 min, then the 0.5% BSA/PBS solution was added to reach 10 mL, and samples were centrifuged at 300 xg at 4 °C for 5 min. Cells were suspended in 1 mL of 0.5% BSA/PBS solution and sorted using a FACS Aria 2 (BD). For further details, see Supplemental Material .
6
Immunophenotyping of Bone Marrow Cells
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Immunophenotyping of bone marrow (BM) was performed using an FC500 or Facscalibur flow cytometer. FACS buffer was made with PBS+2mM EGTA+2% FBS. Primary AML and leukemic stem cell fractions were detected using the following antibodies (company; product #; clone): anti-CD34 PE (BD bio-sciences; 348057; 8G12), anti-CD34 FITC (BD Pharmingen; 555821; 581), anti-CD38 APC (ebiosciences; 17-0389-42; HIT2) and anti-CD123 PE (BD Pharmingen; 558714; 7G3), anti-CD45 APC (BD Pharmingen; 557513; TU116) and anti-class I HLA A, B, C (Biolegend; 311404; W6/32). NK-92 cells lines were assessed for CD16 expression using CD16 PE (Biolegend; 302008; 3G8). Leukemia cell lines were evaluated using anti-CD123 PErCy5.5 (BD Biosciences; 560904; 7G3). Cell sorting was performed using a FacsAria cell sorter as described in the Online Supplementary Methods.
7
Mouse and Human gp100 Peptide Protocol
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Mouse gp10025–33 (mgp100, EGSRNQDWL) and human gp10025–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea). CD8 microbeads were purchased from Miltenyi Biotec (Auburn, AL, USA). All antibodies used for flow cytometry were purchased from BD Bioscience, including anti-Thy1.1-FITC, anti-CD3-FITC, anti-B220-FITC, anti-CD62L-FITC, anti-Ly-6C-FITC, anti-CD19-PE, anti-CD8-PE, anti-CD44-PE, anti-CD8-PE-Cy5, anti-Thy1.1-PE-Cy5, anti-CD11b-PE-Cy5, anti-CD4-APC, anti-CD8-APC, and anti-CD45-APC. Recombinant human IL-2 (Proleukin) was purchased from Novartis, and the Fc fusion protein of human IL-7 (IL7-Fc), which consist of the extracellular domain of human IL-7 (aa 26–177) fused to the N-terminus of the Fc portion of a mutant human IgG1, was obtained from AdipoGen (Seoul, Korea). The CellTrace CFSE Cell Proliferation Kit was purchased from Invitrogen (Carlsbad, CA, USA).
8
Immunophenotyping of Mesenchymal Cell Types
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The immunophenotypes of the BM-DFATs, SC-DFATs, BM-MSCs, ASCs at passage 2 were identified using flow cytometry as previously described [13 (link)]. The cells grown to 60% confluence were suspended at a density of 5 × 105 cells per tube and incubated with various anti-human antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC). The following antibodies were used: anti-CD73-PE, anti-CD90-APC, anti-CD105-PE, anti-CD31-PE, anti-CD45-APC, anti-HLA-DR-PE, anti-CD106-PE, anti-CD54-APC, and anti-CD36-PE (all from BD Biosciences, San Jose, CA). Mouse IgG1-PE, mouse IgG1-APC, mouse IgG2a-PE, mouse IgG2b-APC, and mouse IgM-PE (all from BD Biosciences) were used as negative controls. The fluorescence intensity of the cells was evaluated by a FACSAria flow cytometer (Becton Dickinson, Bedford, NJ), and data were analyzed using FlowJo software (version 10.6.1, FlowJo, Ashland, OR). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes. A minimum of 1 × 104 events were recorded for each sample, and analysis was performed at least three separate times for each condition tested.
9
Multimodal Analysis of Cellular Adhesion Proteins
10
Isolation and Purification of Lung Epithelial Cells
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Whole lungs were harvested from euthanized mice (Cracd WT or Cracd KO) after perfusing 10 ml of cold phosphate-buffered saline (PBS) into the right ventricle. The lung was digested in Leibovitz’s medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington), and 2 mg/mL DNase I (Worthington) at 37 °C for 45 min. The tissue was triturated with a pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5,000 r/min for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5,000 r/min for 1 min, and washed with 1 mL ice-cold Leibovitz’s medium with 10% FBS. In single-cell RNA sequencing (scRNA-seq), digested lung cells were resuspended in 400 μl of buffer with 5 μl of anti-CD31-FITC (BD Biosciences, CA, USA), 5 μl of anti-CD45-APC (BD Biosciences), and 5 μl of anti-CD326 (EpCAM)-PE-Cy7 (Biolegend) and incubated for 30 min at 4 °C. Cells were then washed twice, followed by sorting of the epithelial cells (EpCAM+ / CD31− / CD45−) by fluorescence-activated cell sorting at the Cytometry and Cell Sorting Core at the Baylor College of Medicine.
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