BM collection, LSK staining, and sorting were done as described before[19 (link)]. Briefly, BM was made from either male or female 6 week-old C57BL/6 WT mice. Long bones from all four limbs of each mouse were flushed thoroughly using 27G1/2 needle with 8-10 mL of heparin medium consisting of Hank’s Balanced Salt Solution (HBSS) supplemented with 1 % Penicillin/Streptomycin, and 20 U/mL heparin. Flushed BM cells were layered on top of Ficoll (hydrophilic polysaccharide) and centrifuged at 1500 rpm for 30 min at room temperature. Low-density BM cells were collected at the interface of medium and Ficoll. These low-density BM cells were washed once with stain wash (PBS, 1% bovine calf serum, and 1% penicillin-streptomycin) followed by antibody staining for 15 min on ice. The following cocktail of antibodies was used: phycoerythrin (PE)-conjugated CD3, CD4, CD45R, Ter119, and Gr1; AF700-conjugated c-Kit (CD117, eBioscience); PE-Cy7-conjugated Sca-1; fluorescein isothiocyanate (FITC)-conjugated CD34; allophycocyanin (APC)-conjugated Flk2 (eBioscience). Unless noted otherwise, all monoclonal antibodies were obtained from BD Biosciences. Cells were washed once more with stain wash buffer, and data was acquired using a BD LSRII (BD Biosciences).
Fitc conjugated cd34
Manufactured by Thermo Fisher Scientific
Sourced in United States
FITC)-conjugated CD34 is a fluorescently labeled antibody that binds to the CD34 antigen, which is a cell surface marker expressed on hematopoietic stem and progenitor cells. This product is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2, by BD (2 mentions)
Af700 conjugated c kit cd117, by Thermo Fisher Scientific (2 mentions)
Pe cy7 conjugated sca 1, by Thermo Fisher Scientific (2 mentions)
Allophycocyanin apc conjugated flk2, by Thermo Fisher Scientific (2 mentions)
Cd34 conjugated microbeads, by Miltenyi Biotec (1 mentions)
Automacs separator, by Miltenyi Biotec (1 mentions)
Cellquest pro software, by BD (1 mentions)
Annexin v 7 aad, by BD (1 mentions)
Human cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Interleukin 3 (il 3), by Miltenyi Biotec (1 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (1 mentions)
Granulocyte macrophage colony stimulating factor, by Miltenyi Biotec (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
Ficoll, by Merck Group (1 mentions)
Egm 2, by Lonza (1 mentions)
Pe conjugated cd 133, by Thermo Fisher Scientific (1 mentions)
6 protocols using fitc conjugated cd34
1
Murine Bone Marrow Cell Isolation and Flow Cytometry
2
Purification and Characterization of iPSC-derived EPCs
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In order to exclude the contamination of human iPSCs, the generated EPCs were purified by using MACS according to the manufacture’s instruction. In brief, the differentiated cells (107 cells) were incubated with 10 μl CD34-conjugated microbeads (Miltenyi Biotec Inc) in the refrigerator for 20 min, followed by wash in PBS for twice. The beads-binding cells were separated using an autoMACS separator (Miltenyi Biotec Inc). All CD34+ cells were resuspended with EPC culture medium and cultured in a regular humidified incubator.
For flow cytometry analysis, the purified EPCs were incubated with FITC-conjugated CD34, FITC-conjugated KDR, FITC-conjugated Oct3/4 or FITC-conjugated IgG for 30 min (5 μl, eBioscience) in the dark. All antibodies were purchased from eBioscience. After incubation, all samples were analyzed under flow cytometry (Accuri C6 flow cytometer). 10,000 events were collected for data analysis.
For flow cytometry analysis, the purified EPCs were incubated with FITC-conjugated CD34, FITC-conjugated KDR, FITC-conjugated Oct3/4 or FITC-conjugated IgG for 30 min (5 μl, eBioscience) in the dark. All antibodies were purchased from eBioscience. After incubation, all samples were analyzed under flow cytometry (Accuri C6 flow cytometer). 10,000 events were collected for data analysis.
3
Isolation and Characterization of Murine Hematopoietic Stem Cells
4
Viability of CD34+ Cells with MDS-MVs
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The viability rate was evaluated by FC, after 24hours of co-culture of CD34+ cells with MSC-derived MVs (from 10 MDS patients and 10 controls), using APC H7 Annexin V DY634 (Immunostep #ANXVDY, Salamanca, Spain). After 24hours with and without MVs co-cultured cells were harvested, washed and incubated in the Annexin V binding buffer. Annexin V, 7 AAD (# 51-68981E, BD Biosciences) and FITC-conjugated CD34 (11-0349-42, eBioscience, Inc.San Diego, CA) were added, followed by flow cytometric evaluation. Samples were analyzed on a FACSCalibur flow cytometer using Cellquest Pro software (Becton Dickinson). At least 50,000 events/sample were recorded. Data were analyzed using the Infinicyt program (Cytognos). Viable cells were considered if they were not early or late apoptotic cells (APC H7 Annexin V+/7AAD- and APC H7 Annexin V+/7AAD+, respectively).
5
Isolation and Co-culture of CD34+ Progenitor Cells
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Mobilized CD34+ progenitor cells were isolated from leukapheresis
samples from 5 HD (male/female ratio: 3/2; median age: 34 years, range:
18–54 years) as previously described.18 (link) CD34+progenitor cells were labeled using the human CD34 MicroBead Kit (Miltenyi
Biotec GmbH, Bergisch Gladbach, Germany) and purified in an AUTOMACs device.
The purity and viability of immunomagnetically sorted CD34+ cells
were determined by flow cytometry using fluorescein isothiocyanate
(FITC)-conjugated CD34 (11-0349-42, eBioscience Inc., San Diego, CA, USA)
and 7AAD.
These cells were then co-cultured with MSCs pretreated or not with EP. After
24 h of co-culture, 2 × 103CD34+ were seeded in
methylcellulose semisolid MACS medium supplemented with stem cell factor
(SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF),
granulocyte colony-stimulating factor (G-CSF), IL-3, and IL-6 (Miltenyi
Biotec GmbH, Germany) and maintained for 14 days in a humidified atmosphere
at 37ºC with 5% CO2. CFU-GM colonies were then scored with an
inverted microscope, as previously described.24 (link)
samples from 5 HD (male/female ratio: 3/2; median age: 34 years, range:
18–54 years) as previously described.18 (link) CD34+progenitor cells were labeled using the human CD34 MicroBead Kit (Miltenyi
Biotec GmbH, Bergisch Gladbach, Germany) and purified in an AUTOMACs device.
The purity and viability of immunomagnetically sorted CD34+ cells
were determined by flow cytometry using fluorescein isothiocyanate
(FITC)-conjugated CD34 (11-0349-42, eBioscience Inc., San Diego, CA, USA)
and 7AAD.
These cells were then co-cultured with MSCs pretreated or not with EP. After
24 h of co-culture, 2 × 103CD34+ were seeded in
methylcellulose semisolid MACS medium supplemented with stem cell factor
(SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF),
granulocyte colony-stimulating factor (G-CSF), IL-3, and IL-6 (Miltenyi
Biotec GmbH, Germany) and maintained for 14 days in a humidified atmosphere
at 37ºC with 5% CO2. CFU-GM colonies were then scored with an
inverted microscope, as previously described.24 (link)
6
Isolation and Characterization of Endothelial Progenitor Cells
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Human blood samples were collected in heparin anticoagulant tubes through venipuncture, and peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation with Ficoll (1.077 g/ml; Sigma). At last, PBMC were incubated for 30 min at room temperature in a dark room with appropriate concentrations of the following monoclonal antibodies: Alexa Fluor 647 conjugated KDR (BD Biosciences), FITC conjugated CD34 (eBioscience), PE conjugated CD133 (eBioscience). Appropriate isotype controls were used. Samples were assessed by ow cytometer with FACS Cell Quest analysis software (Becton-Dickinson, CA, USA). At least 1,000,000 events were recorded in the mononuclear cell gate set on the SSC/FSC morphological plot, and the results were analyzed by FlowJo V10 software (Tree Star Inc, Ashland, USA).
Endothelial progenitor cell culture PBMC obtained by density gradient centrifugation were seeded on bronectin (Corning)-coated plates in order to evaluate cell's function. 2 million cells per well were seeded in 24-well plates, and 4 million cells per well were seeded in 12-well plates. Cells were cultured in EGM-2(Lonza), after 4 days, non-adherent cells were removed, and fresh mediums were added. The culture was maintained through day 21, and adherent cells were subjected to further examinations.
Endothelial progenitor cell culture PBMC obtained by density gradient centrifugation were seeded on bronectin (Corning)-coated plates in order to evaluate cell's function. 2 million cells per well were seeded in 24-well plates, and 4 million cells per well were seeded in 12-well plates. Cells were cultured in EGM-2(Lonza), after 4 days, non-adherent cells were removed, and fresh mediums were added. The culture was maintained through day 21, and adherent cells were subjected to further examinations.
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