The colony‐forming potential of hematopoietic stem/progenitor cells was assessed by culture on methylcellulose (H4320, STEMCELL Technologies) supplemented with 1% penicillin/streptomycin (Invitrogen) and cytokines, as described previously.17 BFU‐E, CFU‐Mix, CFU‐GM, CFU‐G, and CFU‐M colonies were assessed on day 14. The lineage differentiation potential of hematopoietic stem/progenitor cells was assayed in IMDM basic medium containing 10% FBS and hematopoietic cytokines (100 ng/ml SCF, 50 ng/ml IL‐6, 20 ng/ml FLT3L, 20 ng/ml IL‐3, 10 ng/ml GM‐CSF, 10 ng/ml G‐CSF, 10 ng/ml TPO, and 4 U/ml EPO). Anti‐human CD45‐APC/Cy7 (BioLegend, Catalog No.: 368516), anti‐human CD71‐FITC (BD Biosciences, Catalog No.: 555536), anti‐human GPA(235a)‐APC (BD Biosciences, Catalog No.: 551336), anti‐human CD66B‐PE (BioLegend, Catalog No.: 305105), and anti‐human CD14‐PE/Cy7 (BD Biosciences, Catalog No.: 557742) were used to detect erythrocytes, granulocytes, and monocytes on day 10 of lineage induction.
Anti human cd45 apc cy7
Manufactured by BioLegend
Sourced in Austria
Anti‐human CD45‐APC/Cy7 is a fluorescence-labeled antibody that binds to the CD45 cell surface antigen, which is expressed on all human leukocytes. This product is intended for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
H4320, by STEMCELL (1 mentions)
Anti human cd71 fitc, by BD (1 mentions)
Anti human cd66b pe, by BioLegend (1 mentions)
Anti human cd14 pe cy7, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Cd3 pacific blue, by BioLegend (1 mentions)
Cyan flow cytometer, by Beckman Coulter (1 mentions)
Anti mouse cd45 percp cy5, by BioLegend (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Cd123 pe, by BioLegend (1 mentions)
Trucount beads, by BD (1 mentions)
Cd14 apc, by BioLegend (1 mentions)
Cd8 pe cy7, by BioLegend (1 mentions)
Hla dr pe cy7, by BioLegend (1 mentions)
Cd4 fitc, by BD (1 mentions)
Anti human mouse gpr91 sucnr1 fitc, by Alomone (1 mentions)
Percp anti mouse cd45, by BioLegend (1 mentions)
Pe anti mouse cd80, by BioLegend (1 mentions)
Apc anti mouse f4 80, by BioLegend (1 mentions)
Anti mouse cd45 efluor450, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti human cd45 apc cy7
1
Hematopoietic Stem Cell Colony Assay
2
Multiparameter Flow Cytometry for Immune Cell Profiling
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Single cell suspensions of BM, liver, lung, spleen, and peripheral blood from engrafted mice were blocked with rat and mouse serum then labeled with Live Dead® Aqua (Life Technologies), anti-mouse CD45 PerCP Cy5.5, anti-human CD45 APC Cy7, HLA DR PE Cy7, CD19/20 Pacific blue and either CD141 APC, CD123 PE (all from BioLegend), and CD1c FITC (Abcam) to identify DC, or CD3 Pacific blue, CD8 PE Cy7, CD14 APC (all from BioLegend), and CD4 FITC (BD Biosciences) for other leukocytes (Figure S1 in Supplementary Material). Absolute cell counts were determined by the addition of 5,000 Trucount beads (BD Biosciences) per tube. Data were acquired on a Cyan flow cytometer (Beckman Coulter) and analyzed using Flow Jo software (Tree star, version 8).
3
Peritoneal Macrophage Immunophenotyping by FCM
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Peritoneal cell pellets collected from the peritoneal fluid were suspended in PBS and stained with the following antibodies: anti-human CD14 -PerCy5.5 (BioLegend,325,621), anti-human CD45 APC-Cy7 (BioLegend, 368,515) and anti-human GPR91/SUCNR1 FITC (Alomone, ASR-090-F). After staining for half an hour, the cells were washed and prepared for FCM (Beckman Coulter). Data were analyzed using FlowJo (v10) software.
Flow cytometry was also performed to analyze the expression of CD80, CD86, CD163, CD206 and MCT1 in macrophages in vivo or THP-1 cells in vitro, as well as SUCNR1 levels in hESCs. The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305,419); anti-human/mouse MCT1 PE (R&D, FAB8275P); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103,129); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD80 PE (BioLegend, 104,707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105,024); anti-mouse CD163 BV421 (BioLegend, 155,309); anti-mouse CD206 BV605 (BioLegend, 141,721), anti-mouse CD11b PCy 7(BioLegend, 101,215); anti-mouse F4/80 APC (BioLegend, 123,116).
Flow cytometry was also performed to analyze the expression of CD80, CD86, CD163, CD206 and MCT1 in macrophages in vivo or THP-1 cells in vitro, as well as SUCNR1 levels in hESCs. The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305,419); anti-human/mouse MCT1 PE (R&D, FAB8275P); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103,129); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD80 PE (BioLegend, 104,707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105,024); anti-mouse CD163 BV421 (BioLegend, 155,309); anti-mouse CD206 BV605 (BioLegend, 141,721), anti-mouse CD11b PCy 7(BioLegend, 101,215); anti-mouse F4/80 APC (BioLegend, 123,116).
4
Wnt3a-Mediated Xenograft Transplantation
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This study was carried out in accordance to the Dutch law on Animal Welfare and Experiments. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Erasmus University Medical Centre Rotterdam, The Netherlands. Intrabone transplantations were performed under isoflurane anesthesia. All animals were housed in groups in individually ventilated cages. Food and water were available ad libitum.
NSG mice were sublethally irradiated (3 Gy) and subsequently transplanted with the progeny generated from 1,00E+05 UCB-derived CD34+ cells cultured in our SFT medium, with or without the addition of 250 ng/ml Wnt3a for 7 days. Each group contained 5 mice. Engraftment was assessed every 2 weeks starting at 3 weeks after transplantation by flowcytometric analysis of the peripheral blood, using a flowcytometric panel including anti-mouseCD45-eFluor450, (eBioscience, Vienna, Austria) and anti-humanCD45-APC-Cy7 (BioLegend, London, UK). Mice were considered engrafted when human CD45 levels were higher than 0.1%. At 17 weeks after transplantation, the mice were sacrificed by cervical dislocation and cells from femurs were analysed.
NSG mice were sublethally irradiated (3 Gy) and subsequently transplanted with the progeny generated from 1,00E+05 UCB-derived CD34+ cells cultured in our SFT medium, with or without the addition of 250 ng/ml Wnt3a for 7 days. Each group contained 5 mice. Engraftment was assessed every 2 weeks starting at 3 weeks after transplantation by flowcytometric analysis of the peripheral blood, using a flowcytometric panel including anti-mouseCD45-eFluor450, (eBioscience, Vienna, Austria) and anti-humanCD45-APC-Cy7 (BioLegend, London, UK). Mice were considered engrafted when human CD45 levels were higher than 0.1%. At 17 weeks after transplantation, the mice were sacrificed by cervical dislocation and cells from femurs were analysed.
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