Kupffer cells were washed with PBS/1% BSA/0.1% NA azide (staining buffer), blocked with Fc block (BDbiosciences, San Jose, CA) for 10 min on ice, incubated with anti-human CD45 (BDbiosciences) for 30 min, followed by intra-cellular staining with anti-human CD68 (clone# Ki-M7, AbD serotec, Raleigh, NC) using BD Cytofix/Cytoperm fixation/permeabilization kits (BDbiosciences). Hepatocytes were washed with staining buffer followed by intra-cellular staining with goat anti-human serum albumin (MyBiosource, San Diego, CA). Approximately 5 million events were acquired for each staining. Peripheral blood (1–2 μl) drawn from the vein of the tail was collected in heparin-coated capillaries (Sigma-Aldrich, St Louis, MO), washed twice with PBS, suspended in PBS/1% BSA, blocked with Fc block for 15 minutes at RT and stained with anti-human CD235 (BDbiosciences) and Retic-Count (BDbiosciences) for 30 minutes. Bone marrow cells were obtained by flushing both tibias as described [21 (link)] and cell surface stained with anti-human CD45, anti-human CD71, anti-human CD235, and anti-mouse Ter-119 antibodies (BDbiosciences). Apoptosis in bone marrow cells was measured using Annexin V/7-ADD apoptosis detection kit (BD Biosciences).
Anti human cd45
Manufactured by BD
Sourced in United States
Anti-human CD45 is a laboratory reagent used for the identification and analysis of human CD45-positive cells. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of all hematopoietic cells. This reagent can be used in flow cytometry and other immunoassay applications to detect and quantify CD45-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd34, by BD (5 mentions)
Anti human cd90, by BD (3 mentions)
Anti human cd44, by BD (3 mentions)
Anti human cd3, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti human cd33, by BD (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Anti human cd11b, by BD (2 mentions)
Anti human cd31, by BD (2 mentions)
Anti human cd14, by BioLegend (2 mentions)
Anti human hla dr, by BD (2 mentions)
Anti human cd19, by BD (2 mentions)
Human serum, by Euroclone (1 mentions)
Nsg mice, by Charles River Laboratories (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Granzyme b, by BD (1 mentions)
Heparin coated capillary tubes, by Thermo Fisher Scientific (1 mentions)
Perforin, by BioLegend (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Annexin 5, by Roche (1 mentions)
22 protocols using anti human cd45
1
Comprehensive Immune Cell Profiling in Liver and Blood
2
Establishment of MLL-AF9 AML Xenografts
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Primary MLL-AF9-expressing cells were obtained from BM samples of diagnosed AML pediatric patients stored in the BioBank of the laboratory of Pediatric Hematology of the University Hospital of Padua, (Italy) according to the guidelines of the local ethics committee. Initial AML xenografts were established by tail vein injection with 8 × 106 primary cells suspended in 300 μl of PBS in 6- to 8-week-old NSG mice, which were purchased from Charles River (Wilmington, MA, USA). All animal experiments were performed in accordance with institutional guidelines and established protocols [53 (link)]. Engraftment was monitored by weekly blood collections and flow cytometry analysis with antihuman CD45 (BD Biosciences). The engraftment rate was defined by the number of days required for the transplanted human CD45+ cells to reach at least 20% in the peripheral blood. Human leukemic cells from the spleens of engrafted mice were collected and cultivated in RPMI supplemented with 10% Human serum (Euroclone), antibiotics, and cytokines SCF, FLT-3L and TPO (40 ng/ml for each), IL-3 and IL-6 (20 ng/ml for each). (All cytokines were obtained from Inalco, Milan, Italy). For ex vivo experiments, two independent biological replicates were performed.
3
Multiparametric Flow Cytometry Analysis
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Blood drawn for the tail vein (50 μl) was collected with heparin-coated capillary tubes (Fisher Scientific, Pittsburgh, PA). Erythrocytes were lysed using ACK lysis buffer (Invitrogen, San Diego, CA) and white blood cells were analyzed by FACS using specific antibodies12 (link). Splenic and thymic cells were isolated as described1 (link)12 (link). Cell populations were quantified by FACS on mononuclear FSC/SSC gating using anti-human CD45 (clone #2D1), CD3 (#HIT3a), CD4 (#SK3), CD8 (#RPA-T8), and CD19 (#H1B19), HLA-DR (#tu39), HLA-A2 (#BB7.2), and intracellularly stained with granzyme B (#GB11) (BD Biosciences, San Diego, CA), perforin (#B-D48, BioLegend, San Diego, CA) or FOXP3 (#PCH101, eBiosiences, San Diego, CA) Abs. HLA-A2/GIL dextramers were obtained from Immudex (Copenhagen, Denmark) and GIL-specific CD8 T cells were enumerated by FACS following the manufacturer’s instructions.
4
Annexin V/PI Flow Cytometry Analysis of Cell Apoptosis
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Cell apoptosis was analyzed by flow cytometry of annexin V (Roche Diagnostics, Indianapolis, IN, USA) and propidium iodide (PI) (Sigma Chemical, St. Louis, MO, USA) positivity on a gated CD45+ population (anti-human CD45, BD Pharmingen, San Diego, CA, USA). To examine and compare inhibitor-specific apoptosis, we calculated % of specific apoptosis as previously described: % specific apoptosis = (tested − control) / (100 − control) × 100, where “tested” is the percentage of annexin V/PI-positivity in treated cells, and “control” is the percentage of annexin V/PI-positivity in untreated cells (spontaneous apoptosis).25 (link)
5
Comprehensive Immune Cell Profiling by Flow Cytometry
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All antibodies used for flow cytometry were mouse anti-human mAbs. Anti-human CD45 (Cat No: 340910), CD56 (Cat No: 345811), CD123 (Cat No: 564195), HLA-DR (Cat No:756414) were purchased from BD Biosciences. Anti-human CD3 (Cat No: 317321), CD19 (Cat No: 302239), CD88 (Cat No: 344304), CD89 (Cat No: 354120), CD14 (Cat No: 325618), CD45RA (Cat No: 304142), CD1c (Cat No: 331520), CD141 (Cat No: 344112) were purchased from BioLegend.
Samples were run on LSRFortessa (BD Biosciences) flow cytometer. Initially, all DCs, including pDCs, cDC1s and cDC2s, were gated based on CD45+CD3-CD56-CD19-CD88-CD89-. Subsequently, pDCs were specifically identified by gating on CD123+CD45RA+. Following the exclusion of pDCs, cDC1s were identified based on the expression of CD141, while cDC2s were identified based on the expression of CD1c. Data were analyzed with FlowJo v.10.8.1.
Samples were run on LSRFortessa (BD Biosciences) flow cytometer. Initially, all DCs, including pDCs, cDC1s and cDC2s, were gated based on CD45+CD3-CD56-CD19-CD88-CD89-. Subsequently, pDCs were specifically identified by gating on CD123+CD45RA+. Following the exclusion of pDCs, cDC1s were identified based on the expression of CD141, while cDC2s were identified based on the expression of CD1c. Data were analyzed with FlowJo v.10.8.1.
6
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.
7
Identifying Lung Macrophage Subsets
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The MACS CD206+ enriched population of lung resident macrophages were incubated with FcR Block (422302; BioLegend) for 5 min and stained at a dilution of 1:50 with one of two panels of directly conjugated antibodies for 30 min at 4°C: anti-human CD45 (563792; BD), CD204 (371906; BioLegend), CD206 (321132; BioLegend), CD14 (562698; BD Biosciences), CD16 (302028; BioLegend), ACE2 (FAB933P; R&D), HLA-DR (307618; BioLegend), CD11b (393114; BioLegend), CD11c (301644; BioLegend); anti-human CD45 (324016; BioLegend), CD204 (371904; BioLegend), and CD206 (321103; BioLegend). Stained cells were then washed with FACS buffer (2% FBS in PBS) three times, and then incubated with cell viability marker propidium iodide (PI, 1 μg/ml, 421301; BioLegend). Flow cytometry was performed on a FACS Aria II (BD Biosciences).
Living (PI−) single, immune (CD45+), and lung resident macrophages (CD206+) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206+CD204hi and CD206+CD204lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.
Living (PI−) single, immune (CD45+), and lung resident macrophages (CD206+) were stained for the above panel of cell surface antigens that have previously been suggested to segregate them into AMs and IMs, and that were differentially expressed according to the scRNA-seq transcriptomic profiles obtained from lung slice culture and sorted into CD206+CD204hi and CD206+CD204lo populations. The sorted populations were directly subjected to 10x single-cell mRNA sequencing at BSL2 as described above, which confirmed their molecular identities as AMs and IMs, respectively.
8
Phenotypic Characterization of Dermal Fibroblasts
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MSC markers in cultured DFCs were analyzed by fluorescent activated cell sorting (FACS) method. (BD FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). DFCs at 80-90% confluence at passage 3, were fixed with 3.7% formaldehyde for 1 h followed by washing thrice with DPBS. The cells were directly labeled with fluorescein isothiocyanate (FITC)-conjugated primary antibodies [anti-mouse CD44 (1:100; BD PharmingenTM, BD Biosciences, Franklin Lakes, NJ, USA), anti-human CD 34 (1:100; BD Biosciences) anti-human CD45 (1:100; BD Biosciences)] or with unconjugated primary antibodies [mouse monoclonal IgG2a CD105 (1:100; Santa Cruz biotechnology, Inc., Dallas, TX, USA) and anti-human CD90 (1:100; BD Biosciences)]. A total of 10,000 cells were analyzed by flow cytometry using CellQuest software (Becton Dickinson).
9
Enrichment and Detection of FGFR2-Positive CTCs
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A total volume of 8 mL of peripheral blood was drawn from a healthy volunteer. A total of 250 cultured tumor cells of each four GC cell lines was spiked into 2 mL of the healthy volunteer's peripheral blood. Peripheral blood mononuclear cell fraction was enriched by Ficol at 1500 g for 15 minutes, followed by dividing into four samples. The sample was stained by propidium iodide (PI; BD Bioscience), anti‐human CD45 (BD Bioscience), anti‐FGFR2 (mFR2, Daiichi Sankyo Co) conjugated with Alexa Fluor 488 (G100L‐Alexa 488), and anti‐EpCAM conjugated with APC (BD Bioscience: 347200). As control, the sample was stained with IgG1 isotype control. Flow cytometry analysis was performed on a FACScan (BD LSR II; Becton Dickinson). When the cutoff value of FGFR2‐positive fluorescence was determined 1000, the FGFR2‐positive number of OCUM‐2MD3 and KATO‐III was high, and the FGFR2‐positive number of MKN45 and NUGC3 was low in the spike test. FGFR2+ cells were determined FGFR2 Alexa488 >103 after the exclusion of PI‐positive and CD45‐positive cells. Isotype negative control was defined as PI‐negative and CD45‐negative to exclude nonspecific binding of the primary antibody.
10
Engineered EGFR-sdCAR Jurkat Cells
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Jurkat cells stably expressing EGFR-sdCARs with varying hinge domains were generated using lentiviral transduction as described below, using an MOI of 1. CAR-expressing cells were then stained with anti-sdAb antibody described below and subjected to cell sorting using a MoFlo Astrios cell sorting flow cytometer (Beckman Coulter, Mississauga, ON, Canada) in order to isolate populations of EGFR-sdCAR-Jurkat cells with similar levels of CAR surface expression. Healthy cultures of EGFR-sdCAR-Jurkat cells were then stained with anti-human CD45 (BD Bioscience, USA, Cat#563717), then resuspended at 5x105 cells per mL in RPMI-complete media. Healthy cultures of SKOV3 or Ramos target cells were then harvested and resuspended in fresh RPMI-Complete media at 5x105 cells per mL. CAR-Jurkat and target cell cultures were then mixed at varying effector to target ratios in a 96-well plate, followed by incubation at 37°C for 30 minutes and immediate assessment via flow cytometry using a BD LSR Fortessa device. The proportion of CAR-target cell doublet formation was assessed as described in the main text. Experiments were repeated 3 times in duplicate.
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