All mouse experiments were approved by the Austrian Ministry of Education, Science and Research (BMBWF-66.010/0056-V/3b/2019). 5x105 CRISPR-modified HSPCs were intrafemorally transplanted into irradiated 8–12-week-old NSG mice. For competitive transplants, 1x105 sort-purified HSPCs (1:1 mixture of CALR INS and WT cells) were intrahepatically transplanted into new-born NSGW41 mice. Human engraftment in murine bone marrow was evaluated after 8, 16 and 24 weeks via flow cytometry. Cells were stained with mTer119-BUV661, mCD45-APC-Cy7, hCD45-BB700, CD33-PE (BD Biosciences), CD19-SB600 (eBioscience, San Diego, CA, USA), CD41-PE-Cy7 (BioLegend) antibodies and SYTOX Red (Invitrogen, Carlsbad, CA, USA) and measured on a CytoFLEX LX flow cytometer (Beckman Coulter).
Cd41 pe cy7
Manufactured by BioLegend
Sourced in United States
CD41-PE-Cy7 is a fluorescently labeled antibody that binds to the CD41 antigen. CD41 is a cell surface protein that is expressed on platelets and megakaryocytes. The PE-Cy7 fluorescent dye is conjugated to the antibody, allowing for the detection and analysis of CD41-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Hcd45 bb700, by BD (2 mentions)
Cd19 sb600, by Thermo Fisher Scientific (2 mentions)
Sytox red, by Thermo Fisher Scientific (2 mentions)
Mcd45 apc cy7, by BD (2 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (2 mentions)
Cd33 pe, by BD (2 mentions)
Cd62 pe, by BioLegend (2 mentions)
Fitc mouse igm k isotype, by BioLegend (2 mentions)
Igg1 k, by BioLegend (2 mentions)
Pac1 fitc, by BioLegend (2 mentions)
Mouse igg1 k isotype, by BioLegend (2 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Ter 119, by Miltenyi Biotec (1 mentions)
Cb 300 k2e, by Sarstedt (1 mentions)
5 protocols using cd41 pe cy7
1
Engraftment of CRISPR-modified HSPCs
2
Platelet Activation Markers Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD41/PECy7 (BioLegend Cat. No. 303718) was used as an identity marker for platelets, PAC-1/FITC (BioLegend Cat. No. 362804) for glycoprotein GP IIbIIIa and CD62/PE (BioLegend Cat. No. 304906) for P-selectin were used as activation markers. IgG1 k (BioLegend Cat. No. 400125), FITC Mouse IgM k Isotype (BioLegend Cat. No. 401605) and Mouse IgG1 k Isotype (BioLegend Cat. No. 400111) were used as isotype control, respectively. The gating strategy of the cell populations was performed according to previously reported by the research group in García-Larragoiti et al. [22 (link)]. Dark conditions and minimal handling were used during the assay to avoid external activation of platelets. Adenosine Di Phosphate (ADP) (20 µM) for 20 min, collagen (20 µM) for 30 min, and epinephrine (EPI) (100 µM) for 40 min were used as positive platelet activation controls [27 (link)]. Concentrations were used following the instructions suggested by the supplier PAR/PAK II® BIO/DATA CORPORATION (Horsham, PA, USA). The acquisition was performed in a CytoFLEX, BECKMAN COULTER® (Brea, CA, USA). Results were analyzed using FlowJo v 10.8.0.
3
Flow Cytometry for Parasitemia Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Parasitemia was measured by blood smear or flow cytometry with similar results. For flow cytometry, 3 μl of tail blood was collected using an EDTA-coated Microvette (CB 300 K2E; Sarstedt) and diluted in 500 ml PBS. The diluted blood was labeled with antibodies directed against Ter119 (fluorescein isothiocyanate [FITC], TER-119, 1/30; Miltenyi Biotec), CD71 (phycoerythrin [PE], C2, 1/300; BD Pharmingen), and CD41‐PE‐Cy7 (MWReg30, 1/100; BioLegend), fixed, and permeabilized with 4% paraformaldehyde (PFA) with 0.6% saponin for 10 min, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining.
4
Platelet Activation Assay by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PRP was obtained by centrifugation, diluted in Tyrodes buffer and incubated for
20 min with CD41/PECy7 (BioLegend Cat. No. 303718) as identity marker,
PAC-1/FITC (BioLegend Cat. No. 362804) and CD62/PE (BioLegend Cat. No. 304906),
were both used as activation markers (glycoprotein αIIbβIII and P-selectin,
respectively). IgG1 k (BioLegend Cat. No. 400125), FITC Mouse IgM k Isotype
(BioLegend Cat. No. 401605) and, Mouse IgG1 k Isotype (BioLegend Cat. No.
400111) were used as isotype control respectively. Subsequently, platelets were
fixed with paraformaldehyde at 4%. Dark conditions and minimal handling were
used during the assay to avoid external activation of platelets. As positive
controls of platelet activation we used known activation agonists ADP, collagen
and epinephrine and the acquisition was performed by flow cytometry as reported
before by our group.10 (link) Results were analyzed using FlowJo v 10.8.0.
20 min with CD41/PECy7 (BioLegend Cat. No. 303718) as identity marker,
PAC-1/FITC (BioLegend Cat. No. 362804) and CD62/PE (BioLegend Cat. No. 304906),
were both used as activation markers (glycoprotein αIIbβIII and P-selectin,
respectively). IgG1 k (BioLegend Cat. No. 400125), FITC Mouse IgM k Isotype
(BioLegend Cat. No. 401605) and, Mouse IgG1 k Isotype (BioLegend Cat. No.
400111) were used as isotype control respectively. Subsequently, platelets were
fixed with paraformaldehyde at 4%. Dark conditions and minimal handling were
used during the assay to avoid external activation of platelets. As positive
controls of platelet activation we used known activation agonists ADP, collagen
and epinephrine and the acquisition was performed by flow cytometry as reported
before by our group.10 (link) Results were analyzed using FlowJo v 10.8.0.
5
Xenograft Model of HSPC Transplantation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All mouse experiments were approved by the Austrian Ministry of Education, Science and Research (BMBWF-66.010/0056-V/3b/2019). 5 × 105 CRISPR-modified HSPCs were intrafemorally transplanted into irradiated 8–12-week-old NSG mice. For competitive transplants, 1 × 105 sort-purified HSPCs (1:1 mixture of CALR INS and WT cells) were intrahepatically transplanted into new-born NSGW41 mice. Human engraftment in murine bone marrow was evaluated after 8, 16, and 24 weeks via flow cytometry. Cells were stained with mTer119-BUV661, mCD45-APC-Cy7, hCD45-BB700, CD33-PE (BD Biosciences), CD19-SB600 (eBioscience, San Diego, CA, USA), CD41-PE-Cy7 (BioLegend) antibodies and SYTOX Red (Invitrogen, Carlsbad, CA, USA) and measured on a CytoFLEX LX flow cytometer (Beckman Coulter).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!