LCLs from a control (LCL-CTL), a sporadic patient (LCL-SALS) and a A382T TDP-43 mutant patient (LCL-TDP382) were incubated with a vitality dye for 15 minutes (Zombie Violet Fixable Viability Kit, BioLegend). Then, LCLs were incubated for 20 minutes with anti-CD19 antibody for B lymphocytes recognition. Cells were fixed and permeabilized using a kit based on saponin permeabilization (Fixation/Permeabilization Solution Kit, BD) as described [69 (link)]. As negative control for R-loop presence, cells were treated with 60 U/ml of ribonuclease H (RNase H, 5.000 units/ml, NEB) using RNase H buffer at 37°C for 1 hour. As negative control for ssRNAs, cells were treated with 100 μg/mL ribonuclease A (RNase A, 10 mg/mL, Thermofisher) in 0.3M NaCl buffer at 37°C for 1 hour. At the end, cells were stained for one hour with conjugated anti-S9.6 antibody (PE/R-Phycoerythrin Conjugation Kit, Abcam) and analysed by flow cytometry (BD FACS Canto II). Logarithmic amplification was used for all channels and FACSDIVA was used for the analysis. Moreover, SH-SY5Y transfection efficiency was analysed through flow-cytometry, detecting cellular GFP signal.
Pe r phycoerythrin conjugation kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The PE/R-Phycoerythrin Conjugation Kit is a laboratory tool used for the conjugation of R-phycoerythrin to target molecules. R-phycoerythrin is a fluorescent protein derived from red algae, commonly used as a labeling agent in various bioanalytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cytoflex s cytometer, by Beckman Coulter (2 mentions)
Zombie violet fixable viability kit, by BioLegend (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd8 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd4 apc, by Thermo Fisher Scientific (1 mentions)
Cd19 apc h7, by BD (1 mentions)
Ly 6g gr 1 pe cy7, by Thermo Fisher Scientific (1 mentions)
Anti human cd3 percp, by BD (1 mentions)
Nk1.1 pe, by Thermo Fisher Scientific (1 mentions)
Cd14 fitc, by Thermo Fisher Scientific (1 mentions)
Nkp46 pe cy7, by BD (1 mentions)
Cd19 apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Cd11b fitc, by Thermo Fisher Scientific (1 mentions)
Cd11c pe, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Quantibrite pe phycoreythrin fluorescence quantitation kit, by BD (1 mentions)
6 protocols using pe r phycoerythrin conjugation kit
1
R-loop Detection in Lymphoblastoid Cell Lines
2
Phenotyping of Immune Cells in Periodontitis
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PBMCs and periodontal-infiltrating leukocytes from enrolled subjects were surface-stained with anti-human CD3-PerCP (BD Biosciences, San Jose, CA), CD19-APC-H7 (BD Pharmingen), NKp46-PE-Cy7 (BD Pharmingen), and CD14-FITC (eBioscience, San Diego, CA), and were intracellularly stained with anti-human IL-18 (Abcam), which was conjugated by PE/R-Phycoerythrin Conjugation Kit (Abcam). PBMCs, splenocytes, and periodontal-infiltrating leukocytes from mice were surface-stained with anti-mouse CD3-FITC, CD4-APC, CD8-PerCP Cy5.5, NK1.1-PE, CD11b-FITC, CD11c-PE, CD19-APC, and/or Ly-6G (Gr-1)-PE Cy7 (eBioscience) for detection of leukocyte subsets. Periodontal ligament cells were stained with Annexin V-FITC and propidium iodide. Data were acquired using FACS Aria II flow cytometer (BD Biosciences) and were analyzed using FlowJo Version 10 (Tree Star, Ashland, OR).
3
Quantification of Surface MSLN Expression
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Anti-human mesothelin antibody (Clone MB, Catalog No. 530203, BioLegend; San Diego, CA, USA) was conjugated with PE/R-phycoerythrin conjugation kit (Abcam, Cambridge, MA, USA; Catalog No. ab102918) per the manufacturer’s instructions. Samples stained with the PE-conjugated anti-MSLN antibody were analyzed on a NovoCyte 3000 Flow Cytometer. MSLN cell surface expression was quantitated using a BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit (BD Biosciences) following the manufacturer’s protocol.
4
Quantifying GPC2 Surface Expression on Cells
5
Intracellular Brucellae Detection in PBMCs
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To explore the capacity of mAb to recognize intracellular Brucellae in PBMCs of patients with brucellosis, the purified mAb 6C12 was labeled with phycoerythrin (PE) by PE/R-Phycoerythrin Conjugation Kit according to the manufacturer's instructions (Abcam, Cambridge, United Kingdom). FITC-Anti-CD14 antibody (M5E2 clone; BD, Bioscience, USA) was used to recognize the lineage of mononuclear cells. PBMCs collected from 28 brucellosis patients and 55 non-Brucella-infected blood donors were stained for intracellular Brucellae by FACSCalibur flow cytometer with FITC-Anti-CD14 antibody (M5E2) and PE-6C12 specific to FCM Omp25 (Delaporte et al., 2008 (link); Okumura et al., 2015 (link)). Briefly, PBMCs were treated with 5 μl FITC-M5E2 for 30 min at room temperature and cells were washed twice with sample buffer [PBS containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, Missouri, USA)]. After fixation and permeabilization with Intracellular Fixation & Permeabilization Buffer (eBioscience, California, USA), PBMCs were incubated with PE-6C12 for 45 min at 4°C and washed three times. Finally, 300 μl of resuspended PBMCs were tested by FCM. All steps were performed in the dark. The data were analyzed by FlowJo software version v10.0. The cutoff for non-Brucella-infected PBMCs from healthy blood donors was established as 1% by FCM.
6
GPC2 Surface Expression Quantification by Flow Cytometry
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For GPC2-directed flow cytometry, dissociated single cell suspensions were achieved with 0.02% EDTA in HBSS and cells were stained with LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen) for 30 minutes in the dark on ice, washed with cold PBS, incubated in the dark on ice for 30 minutes with the D3-GPC2-IgG1-PE antibody in 10% γ-Globulins (from human blood, Sigma-Aldrich, G4386), washed with cold PBS x 2, and fixed in 1% formaldehyde. Stained samples were run on a Beckman CytoFLEX S cytometer and analyzed using FlowJo software. To semiquantitate GPC2 cell surface expression, a BD Quantibrite Beads PE Phycoerythrin Fluorescence Quantitation Kit (#340495) was run in parallel with samples according to the manufacturer's instructions. The D3-GPC2-IgG1 antibody was conjugated to PE using a PE/R-Phycoerythrin Conjugation Kit (Abcam, ab102918).
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