Heparinized whole blood was used for flow cytometry using the following antibody cocktail: CD45-PerCP (Clone 30-F11; Biolegend 103130), CD3-eFLUO450 (Clone 17A2; eBioscience 48-0032), NK1.1-PE (Clone PK136; BD557391), Ly6G-APC-CY7 (Clone 1A8; BD560600), CD11b PE-CY7 (Clone M1/70; BD552850), Ly6C-APC (Clone 1G7.G10; Miltenyi 130-093-136), CD19-APC-H7 (Clone 1D3, eBioscience 47-0193) and Siglec-F-PE (Clone E50-2440; BD552126). Further, cells from the whole brain were isolated for FACS. Anesthetized mice were perfused with PBS to remove the peripheral blood. Subsequently, brains were dissected, mechanically dissociated and digested with a collagenase mix (including collagenase IX, collagenase I, DNAse and RPMI-HEPES). Immune cells were separated using a Percoll-gradient and stained with CD45 PerCP (Clone 30-F11; Biolegend 103130), CD11c PE-Cy7 (Clone N418, eBioscience 25-0114)), F4/80 (Clone BM8; Biolegend 123116), CD11b BV510 (Clone M1/70; Biolegend 101245), CD3 BV421 (Clone 17A2; eBioscience 48-0032-82), CD19 BV421 (Clone 6D5; Biolegend 115538), Ly6G BV421 (Clone eBio927; eBioscience 48-3172), MHC II-APC-eFluor780 (Clone M5/114.15.2; eBioscience 47-5321-82). All samples were measured with a FACS-Canto II (BD Biosciences). Results were analysed with FACSdiva version 8 (BD Biosciences).
Cd45 percp
Manufactured by BioLegend
Sourced in United States
CD45-PerCP is a fluorescently-labeled monoclonal antibody that binds to the CD45 protein, a common leukocyte antigen expressed on the surface of various hematopoietic cells. The PerCP (Peridinin Chlorophyll Protein) fluorophore is attached to the antibody, allowing for detection and identification of CD45-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Cd3 fitc, by BioLegend (6 mentions)
Cd11b fitc, by BioLegend (5 mentions)
Lsrfortessa, by BD (3 mentions)
F4 80 apc, by BioLegend (3 mentions)
Cd3 apc, by BioLegend (3 mentions)
Cd4 pe, by BioLegend (3 mentions)
Facsaria 3, by BD (3 mentions)
Cd86 pe, by BioLegend (3 mentions)
Ifn γ apc, by BioLegend (3 mentions)
Facscanto 2, by BD (2 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (2 mentions)
Siglecf pe, by BD (2 mentions)
Foxp3 pe, by BioLegend (2 mentions)
Cd34 apc, by BioLegend (2 mentions)
Facscalibur, by BD (2 mentions)
Cd4 apc, by BioLegend (2 mentions)
Cd3 pe, by BioLegend (2 mentions)
Cd8a pe cy7, by BioLegend (2 mentions)
Cd19 fitc, by BioLegend (2 mentions)
Cd8 apc, by BioLegend (2 mentions)
44 protocols using cd45 percp
1
Multiparametric Flow Cytometry of Immune Cells
2
Flow Cytometry Analysis of Cell Surface Markers
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To detect the expression of cell surface markers, the differentiated cells were collected and suspended in FACS buffer (phosphate-buffered saline (PBS), 10 mmol·L−1 EDTA, and 2% FBS) at a density of 2 × 106 cells per ml. Cells were incubated with fluorescent-dye-conjugated antibodies for 30 min on ice in the dark. After being washed three times in FACS buffer, the cells were suspended in PBS. Sorting gates were established based on comparisons with isotype negative controls and compensation controls. Finally, the purified cells were collected into serum-free DMEM media for mRNA expression analysis or into complete media for cell propagation in vitro. The following conjugated antibodies (all from Biolegend) were used in this study: PerCP-CD45 (Cat No.: 368506), APC-CD73 (Cat No.: 344006), FITC-CD146 (Cat No.: 361012), and PE-CD271 (Cat No.: 345106) as previously described.16 (link)
3
MSU Crystals Induce Inflammatory Responses
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MSU crystals were purchased from InvivoGen (San Diego, CA, USA) and used freshly for in-vitro treatment by dissolving in phosphate buffered saline (PBS). Antibodies for cleaved caspase-1 (p20), phospho-NF-κB p65 (ser536), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Human and mouse mature IL-1β were measured using the IL-1β ELISA kit (Neobioscience, Shenzhen, China). PerCP-CD45, PE-F4/80, and APC-Gr-1 antibodies were obtained from BioLegend (San Diego, CA, USA).
4
Multiparametric Flow Cytometry Analysis
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The following antibodies directed against mouse antigens were used: Percp-CD45 (Biolegend 103130), FITC-CD31 (BD 553372), APC-CD117/Kit (BD 553356), PE-CD31 (Biolegend 102407), FITC-Ter119 (BD 561032), Percp-IgG2bk (Biolegend 400336), FITC-IgG2ak (BD 553929), APC-IgG2bk (BD 556924), PE-IgG (Biolegend 405307), PE-Annexin V (BD560930), DAPI (ThermoFisher D1306), PE-ItgαV (Biolegend 104106), and PE-Itgα4 (BD 557420). For flow analysis, antibodies were used at a concentration of 2 µg ml−1.
5
Evaluating Immunological Effects of BayK
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To evaluate potential immunological effects of BayK administration, splenocytes were treated with BayK in vitro. A C57/Bl6 spleen was processed through a 0.7-μm cell strainer and treated with ACK lysis buffer to lyse red blood cells. Splenocytes were plated at 1 million cells per well in a 96-well plate, with 150 μl of RPMI medium. Splenocytes were treated with BayK (0.9 μg/ml; 2% of the in vivo dose and a feasible resulting LN concentration), PMA (20 ng/ml), and ionomycin (1 μg/ml; a positive control known to result in T cell activation) or DMSO (vehicle control). After 4 hours of incubation at 37°C, cells were washed and treated with 2.4G2 Fc block for 5 min on ice. Cells were then stained with the Zombie Aqua Fixable Viability Kit (BioLegend, San Diego, CA) for 30 min at room temperature and then stained for 30 min on ice with a panel of antibodies, including PerCP CD45, BV711 CD3, phycoerythrin (PE)/Cy7 CD4, fluorescein isothiocyanate CD8, Alexa Fluor 700 CD25, BV421 CD69, Alexa Fluor 647 PD-1, and PE FoxP3, all from BioLegend. Cells were then fixed by 15-min incubation in 2% paraformaldehyde on ice and analyzed on a BD LSRFortessa flow cytometer.
6
Pluripotency and NK Cell Induction Analysis
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For analysis of pluripotency markers, iPSCs were dissociated into single cells using TrypLETM Select (Gibco), blocked with 10% human AB serum, stained with Alexa Fluor 488-SSEA-3 and Alexa Fluor 647-TRA-1-60 (BioLegend, San Diego, CA, USA), and analyzed by BD FACS Canto flow cytometer (BD Biosciences) with FlowJo software (V10.4.1). For phenotypic analysis of HPC induction and NK commitment, the cells were resuspended in FACS buffer (PBS with 2% bovine serum albumin), incubated with antibody cocktail for 30 min at 4 °C in the dark, and analyzed by BD FACS Canto flow cytometer. The antibodies used in this study included FITC-CD16, FITC-Nkp46, PE-CD56, PE-Nkp44, PerCP-CD45, PerCP/Cy5.5-CD94, APC-CD34, PE/Cy7-CD43, PE/Cy7-KIR, Alexa Fluor 700-CD161, and BV605-Nkp30 (BioLegend).
7
Murine Monocyte Immunophenotyping by Flow
8
Prostate Cell Isolation and Characterization
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Protocols were adopted from our previous study12 (link). Prostate tissues were processed as described above for bPSC isolation and isolated cells were stained with the following fluorescence conjugated antibodies (Biolegend): CD45-PerCP (#103130), Sca-1-APC (#122512) and CD49f-PE (#313612) before fixation with 10% neutral buffered formalin. To detect AR protein, fixed samples were stained with unconjugated rabbit anti-AR (#ab52615, Abcam, 1:50) followed by secondary stain with Alexa Fluor 488 (Invitrogen, 1:500). Prostate cells isolated from lineage tracing mice were stained with the following fluorescence conjugated antibodies (Biolegend): EpCAM-PE/Cyanine7 (#18215) and CD49f-BV421(#313623). Flow cytometry analyses were performed using BD LSRFortessa.
9
Corneal Flow Cytometry Analysis
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Mouse eyeballs were collected (n = 5) at 3 days p.i., and the corneas were dissected around the scleral–limbal region. The protocol of corneal flow cytometry has been previously described.34 The gate was set on the CD45+ population. The primary antibodies used in this experiment were CD45‐PercP, CD11b‐fluorescein isothiocyanate and Ly6G‐PE (BioLegend, San Diego, CA, USA).
10
Murine PBMC Isolation and Staining
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Blood was collected in a tube with 3.8% w/v sodium citrate. Mouse PBMCs were purified from whole blood by Ficoll (PanEco, Moscow, Russia) density centrifugation. Murine PBMCs staining was performed using the following monoclonal antibodies: CD45-PerCP (103130; BioLegend, San Diego, CA, USA), CD3-PE (100308; BioLegend, San Diego, CA, USA), CD8a-PE/Cy7 (100722; BioLegend, San Diego, CA, USA), CD4-APC (100412; BioLegend, San Diego, CA, USA), CD19-FITC (152404; BioLegend, San Diego, CA, USA), and CD25-Pacific Blue (102022; BioLegend, San Diego, CA, USA).
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