TA muscle was damaged and treated as described above for the ex vivo phagocytosis. After muscle digestion, non‐viable cells were labeled with Ghost Dye Red 780 (Tonbo Biosciences, 13‐0865‐T100, 1:1,000). Then, the cell suspension was incubated with anti‐mouse FcR Blocking Reagent and further stained with BV510‐conjugated anti‐CD45 (Biolegend, 103138, 1:100), AF647‐conjugated anti‐CD64 (Biolegend, 139322, 1:50), PE‐Cy7‐conjugated anti‐Lyve1 (Novus Biologicals, NBP1‐43411PECY7, 1:100), PE‐conjugated anti‐Folate receptor (Biolegend, 153304, 1:100) or AF488‐conjugated anti‐CD206 (eBioscience, 53‐2061‐82, 1:100) antibodies for 20 min at 4°C. Macrophages were analyzed with a FACSCanto II flow cytometer (BD Biosciences).
Bv510 conjugated anti cd45
Manufactured by BioLegend
Sourced in United States
BV510-conjugated anti-CD45 is a flow cytometry antibody that binds to the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of all leukocytes.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (1 mentions)
Ghost dye red 780, by Cytek Biosciences (1 mentions)
Anti mouse fcr blocking reagent, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Fitc conjugated anti cd3, by BioLegend (1 mentions)
Anti cd19, by BioLegend (1 mentions)
Viakrome 808 fixable viability dye, by Beckman Coulter (1 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
3 protocols using bv510 conjugated anti cd45
1
Muscle Macrophage Phenotyping by Flow Cytometry
2
Cryopreserved PBMC Analysis by Flow Cytometry
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On the same day of clinical and ultrasound assessment, we obtained fresh whole blood, isolated peripheral blood mononuclear cells (PBMCs) by Ficoll-Paque Premium (GE Healthcare, Chicago, IL, USA), and stored them in CELLBANKER 2 freezing medium (Nippon Zenyaku Kogyo, Fukushima, Japan) at –80°C. We analyzed the samples by flow cytometry using the following antibodies for surface staining: FITC-conjugated anti-CD3 (UCHT1), anti-CD14 (HCD14), anti-CD19 (HIB19), anti-CD11c (3.9), anti-FcεRIα (AER-37 [CRA-1]), anti-CD94 (DX22), anti-CD123 (6H6), anti-CD34 (581), anti-CD16 (3G8), BV510-conjugated anti-CD45 (HI30), BV421-conjugated anti-CD127 (A019D5), PE-Cy7-conjugated anti-CD117 (c-kit) (104D2), Alexa Fluor 647-conjugated anti-CRTH2 (BM16), and APC-Cy7-conjugated anti-CD56 (HCD56) (All antibodies were from BioLegend, San Diego, CA, USA).
Data were acquired on FACS CANTO II (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Total ILC, ILC1, ILC2, and ILC3 were identified using previously reported gating methods [36 (link)] (S1 Fig ).
We confirmed no significant differences in any populations tested before and after freezing.
Data were acquired on FACS CANTO II (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Total ILC, ILC1, ILC2, and ILC3 were identified using previously reported gating methods [36 (link)] (
We confirmed no significant differences in any populations tested before and after freezing.
3
Quantification of CD64 Expression in Synovial Cells
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Single cells from digested synovium were incubated with Fc-Gamma II Block (1:100, BD Pharmingen, Franklin Lakes, NJ, US) diluted in PBS with 1% FCS and 2 mM EDTA for 15 min, followed by 15 min antibody staining with BV510-conjugated anti-CD45 (1:200, clone HI30, Biolegend, San Diego, CA, US) and PE-conjugated anti-CD64 (1:200, Clone 10.1, Biolegend, San Diego, CA, US). In addition, cells were washed twice with PBS and stained with Viakrome 808 Fixable Viability Dye (Beckman Coulter, Pasadena, CA, US) to assess cell viability. Samples were measured on a CytoFLEX LX Flow Cytometer (Beckman Coulter, Pasadena, CA, US) and analysed using Kaluza Analysis Software (Beckman Coulter Life Sciences, Woerden, NL). Cell debris and doublets were excluded using forward scatter and side scatter and dead cells were removed from the analysis based on eFluor 780 positivity. The CD64 expression for each patient was quantified as the mean fluorescent intensity (MFI) of the CD64 signal in the CD45+ population.
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