Journal: Nature protocols

Article Title: Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells.

doi: 10.1038/nprot.2010.184

Figure Lengend Snippet: Figure 1 | Schematic diagram of the protocol used to obtain multipotent lin–CD34 + CD43 + CD45 + progenitors from human pluripotent stem cells. (a–b) Undifferentiated hiPSCs (iPS (foreskin)-1; a) cocultured with overgrown OP9 (b). (c) After 8 d of coculture, differentiated blood-forming hPSC colonies with radial sac-like structures are formed. (d,e) Typical flow cytometric analysis of differentiated iPS (foreskin)-1 cells collected after 8 d of OP9 coculture shows that hiPSC-derived CD34 + cells consist of CD31 + CD43 − endothelial cells, CD43 + hematopoietic cells and CD31 − CD43 − mesenchymal cells. Three major subsets of hematopoietic progenitors are defined within the CD43 + population: CD235a/41a + erythromegakaryocytic progenitors, and CD235a/CD41a–CD45 − and CD235a/CD41a–CD45 + multipotent progenitors. For the analysis, live (7-AAD–) and human (TRA-1-85 + ) cells are gated. (f) When differentiated hiPSCs are collected on day 8 of OP9 coculture by enzymatic digestion and cultured in nonadherent conditions in the presence of GM-CSF, cells spontaneously form large aggregates, with hematopoietic cells released from these aggregates into suspension. (g) Typical flow cytometric analysis of cells cultured with GM-CSF for 2 d shows a marked expansion of the CD45 + population of hematopoietic progenitors within the CD43 + population. (h–i) After Percoll separation (h) and magnetic sorting (i), a population of CD45 + progenitors with > 90% purity can be obtained. (j) Isolated CD45 + progenitors do not express major lineage markers, but retain expression of CD34, CD90 and CD117 markers, characteristic of primitive hematopoietic cells. Scale bar, 300 µm.

Article Snippet: Cell strainer (40 μm; BD Biosciences, cat. no. 352340) Cell strainer (70 μm; BD Biosciences, cat. no. 352350) MACS separation columns, LD (Miltenyi Biotec, cat. no. 130-042-901) MACS separation columns, LS (Miltenyi Biotec, cat. no. 130-042-401) • • • • table 2 | Antibodies used to analyze differentiation of hematopoietic progenitors and myeloid lineages from hPSCs. name Fluorochrome clone company cat. no. Anti-human CD1a PE VIT6b CALTAG-Invitrogen MHCD1a04 Anti-human CD2 FITC RPA-2.10 BD Biosciences 555326 Anti-human CD3 FITC SK7 BD Biosciences 349201 Anti-human CD7 PE M-T701 BD Biosciences 555361 Anti-human CD10 PE HI10a BD Biosciences 555375 Anti-human CD11b FITC VIM12 CALTAG-Invitrogen CD11b01 Anti-human CD13 PE TüK1 CALTAG-Invitrogen MHCD1304 Anti-human CD14 FITC M5E2 BD Biosciences 555397 Anti-human CD15 FITC VIMC6 Miltenyi Biotec 130-081-101 Anti-human CD16 PE 3G8 CALTAG-Invitrogen MHCD1604 Anti-human CD19 PE HIB19 BD Biosciences 555413 Anti-human CD34 APC 581 BD Biosciences 555824 Anti-human CD41a PE HIP8 BD Biosciences 555467 Anti-human CD43 FITC 1G10 BD Biosciences 555475 Anti-human CD45 APC HI30 BD Biosciences 555485 Anti-human CD64 FITC 10.1 CALTAG-Invitrogen CD6401 Anti-human CD66b FITC G10F5 BD Biosciences 555724 Anti-human CD90 (Thy-1) APC 5E10 BD Biosciences 559869 Anti-human CD115 PE 61708 R&D Systems FAB329P Anti-human CD117 APC YB5.B8 BD Biosciences 550412 Anti-human CD123 FITC AC145 Miltenyi Biotec 130-090-897 Anti-human CD163 PE 215927 R&D Systems FAB1607P Anti-human CD235a PE GA-R2(HIR2) BD Biosciences 555570 DC-SIGN FITC DCN46 BD Biosciences 551264 HLA-DR PE Tü36 CALTAG-Invitrogen MHLDR04 Lactoferrina PE 3C5 CALTAG-Invitrogen GIC206 Langerina PE 343828 R&D Systems FAB2088P Major basic proteina None AHE-2 BD Biosciences 550843 Myeloperoxidasea FITC H-43-5 CALTAG-Invitrogen GIC205 Anti-human TRA-1-85 APC TRA-1-85 R&D Systems FAB3195A aIntracellular staining.

Techniques: Derivative Assay, Cell Culture, Suspension, Isolation, Expressing

Journal: Genes

Article Title: Hp1-1 as a Genetic Marker Regulating Inflammation and the Possibility of Developing Diabetic Complications in Patients with Type 2 Diabetes—Cohort Studies

doi: 10.3390/genes11111253

Figure Lengend Snippet: Effect of haptoglobin phenotype on serum soluble form of CD163 receptor (sCD163) ( A ) and interleukin 10 (IL-10) ( B ) concentration in patients with type 2 diabetes. The sCD163 concentration is expressed as ng/mL serum ± interquartile range (IQR). The IL-10 concentration is expressed as pg/mL serum ± IQR. * p < 0.001—statistical significance between Hp1-1 (control group) and Hp2-1 or Hp2-2. p > 0.05—no statistically significant differences between Hp2-1 and Hp2-2. Hp1-1—haptoglobin phenotype 1-1; Hp2-1—haptoglobin phenotype 2-1; Hp2-2—haptoglobin phenotype 2-2.

Article Snippet: The levels of sCD163 in serum were determined by an enzyme-linked immunosorbent assay (Human CD163 Quantikine ELISA Kit, R&D System, Abingdon, UK) according to the manufacturer’s procedure.

Techniques: Concentration Assay, Control

Journal: Genes

Article Title: Hp1-1 as a Genetic Marker Regulating Inflammation and the Possibility of Developing Diabetic Complications in Patients with Type 2 Diabetes—Cohort Studies

doi: 10.3390/genes11111253

Figure Lengend Snippet: The proposed biological mechanism explaining the relationship between the haptoglobin phenotype and specific markers regulating inflammatory processes. In individuals with type 2 diabetic and Hp1-1 phenotype haptoglobin, hemoglobin (Hb) released intravascularly from red blood cells (RBC) is rapidly bound by haptoglobin (Hp1-1) protein to form an Hb–Hp1-1 complex that is cleared by scavenger receptor CD163 (on type anti-inflammatory macrophage M2). As a result, we obtain a protective effect through the increased production of anti-inflammatory (IL-10) and antioxidant factors. In people with type 2 diabetes and haptoglobin with the Hp2-1 and Hp2-2 phenotype, CD163 clearance is impaired and leads to increased oxidative stress. This results in an enhanced production of inflammatory factors which lead to destruction of the body’s tissues by apoptosis and necrosis of cells. It is known that chronic oxidative stress and the activity of pro-inflammatory factors may intensify the development of complications in people with type 2 diabetes, such as retinopathy, nephropathy, ischemic heart disease, atherosclerosis, stroke, and heart attack.

Article Snippet: The levels of sCD163 in serum were determined by an enzyme-linked immunosorbent assay (Human CD163 Quantikine ELISA Kit, R&D System, Abingdon, UK) according to the manufacturer’s procedure.

Techniques: Activity Assay

Journal: Veterinary research

Article Title: Differential infection behavior of African swine fever virus (ASFV) genotype I and II in the upper respiratory tract.

doi: 10.1186/s13567-023-01249-8

Figure Lengend Snippet: Figure 2 Identification of ASFV infected cells in the nasal explants. A Double IF staining for ASFV p72 (green) in combination with CD163 (red) was performed on the nasal septum (top) and the turbinate (bottom) explant at 72 hpi. Blue color presents the nuclei staining. The dotted yellow lines present a cluster of infection. The dotted white lines indicate the border between the mucosal epithelium (EP) and the lamina propria (LP). ASFV+CD163+ cells and ASFV+CD163− cells are indicated with white arrows and yellow arrows, respectively. Scale bar: 50 μm. B Kinetic study of ASFV infection in the nasal explant. The total number of infected cells in a section from 0, 24, 48, and 72 hpi is presented in mm2 area. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison post hoc test. Different letters represent significant differences (p < 0.05) between time points. All data are presented as mean value of three animals ± standard deviation (SD). C Percentage of ASFV infected cells by the areas in the nasal explants. In both nasal tissue types and genotypes, most infected cells were found in the lamina propria. EP: epithelium, LP: lamina propria, SM: submucosa.

Article Snippet: For the additional characterization of the infected cells, cells were stained by a triple immunofluorescence with a FITC-conjugated ASFV p72 antibody, a human CD163 (R&D systems, Mineapolis, MN, USA) antibody, and a porcine vimentin antibody.

Techniques: Infection, Staining, Comparison, Standard Deviation

Journal: Toxicologic Pathology

Article Title: Biocompatible Solutions: Evaluating the Safety of Repeated Intra-Articular Injections of pMPCylated Liposomes for Knee Osteoarthritis Therapy in Rat Models

doi: 10.1177/01926233241271400

Figure Lengend Snippet: Antibodies used for the immunofluorescence study.

Article Snippet: Novus (CD163) , NBP3-07981 (CD163).

Techniques: Immunofluorescence, Marker