Whole fresh heparinized blood samples or PBMCs were stained with anti-CD123-PE (clone 7G3), anti-HLA-DR-PerCP (clone L243), anti-CD11c-APC (clone B-Ly6) and anti-Lin cocktail-1-FITC (CD3, CD14, CD16, CD19, CD20 and CD56) (B-D BioScience, San Diego, CA). The Lin− HLA-DR+ CD123+ cells (pDCs) as well as Lin− HLA-DR+ CD11c+ (mDCs) were examined by flow cytometry (FACScaliber), and the data were analyzed using FCS Express Software (Vancouver, Canada). In some experiments, the cells were stained with anti-CD40-PE (clone 5C3), anti-CD80-PE (clone L307.4) and anti-CD86-PE (clone 2331). The absolute number of DCs was calculated as follows: absolute mDC or pDC number = (%of mDCs or pDCs in white blood cells obtained by flow cytometry) × (total number of white blood cells per ml blood counted manually by hemacytometer).
Anti hla dr percp clone l243
Manufactured by BD
Sourced in United States
Anti-HLA-DR-PerCP (clone L243) is a fluorochrome-conjugated monoclonal antibody that binds to the human HLA-DR antigen. It can be used for the identification and enumeration of HLA-DR-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (2 mentions)
Anti cd16 pe clone 3g8, by BD (2 mentions)
Anti cd11c apc clone b ly6, by BD (1 mentions)
Granulocyte macrophage colony stimulating factor, by Miltenyi Biotec (1 mentions)
Anti cd14 fitc clone mφp9, by BD (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
Pancoll human, by PAN Biotech (1 mentions)
Flow cytometer facs canto, by BD (1 mentions)
Anti cd45 apc clone 2d1, by BD (1 mentions)
Edta vacutainer, by BD (1 mentions)
Anti cd14 fitc clone m5e2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
Hepes, by Harvard Bioscience (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
5 protocols using anti hla dr percp clone l243
1
Quantifying Dendritic Cell Subsets
2
Isolation and Characterization of Monocyte-Derived Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque™ Plus; GE Healthcare) of buffy coat preparations from anonymous donors (Institute of Transfusion Medicine, Ulm University). Monocytes were isolated from PBMCs by adherence on tissue culture treated plastic flasks (Falcon, Corning, NY, United States). Monocyte-derived macrophages were generated by incubation with granulocyte-macrophage colony-stimulating factor (10 ng/ml; Miltenyi Biotec, Bergisch Gladbach, Germany) in Macrophage-Serum Free Medium (M-SFM; Gibco, ThermoFisher Scientific, Schwerte, Germany) for 6 days. Phenotypic characterization by flow cytometry demonstrated that macrophages expressed CD68 (anti–CD68-FITC, clone Y1/82A; BD Biosciences, Franklin Lakes, NJ, United States) and MHCII (anti–HLA-DR-PerCP, clone L243; BD Biosciences) as described (Bruns et al., 2012 (link)).
3
Isolation of Monocyte Subsets from SpA Patients
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monocyte subpopulations were isolated from peripheral blood mononuclear cells (PBMC) obtained from SpA patients. PBMC were isolated from EDTA-treated whole peripheral blood by the standard Pancoll human (PAN-Biotech, Aidenbach, Germany) density gradient centrifugation. PBMC were washed in PBS (Sigma-Aldrich, Saint Louis, USA) and then monocyte subsets (classical—CD14++CD16−, intermediate—CD14++CD16+ and non-classical—CD14+CD16++) were isolated using flow cytometry cell sorting. The following monoclonal antibodies (mAbs) were used to stain monocytes: anti-CD14-FITC (clone MφP9, BD Biosciences, San Jose, CA, USA), anti-CD16-PE (clone 3G8, BD Biosciences) and anti-HLA-DR-PerCP (clone L243, BD Biosciences), in 1:25 dilution v/v stained and gated as previously described by us and others47 (link),48 (link). The stained monocytes were then incubated for 30 min at 4 °C after which they were sorted using the FACSAria II cell sorter (BD Biosciences). Sorter was equipped with 488 nm laser for excitation of FITC, PE and PerCP. The following band-pass filters were used for the measurement of fluorescence: 530/30 for FITC, 582/42 for PE and 695/40 for PerCP. After isolation, the cells were washed in PBS, centrifuged for 10 min at 350×g and kept frozen at − 80 °C until RNA isolation.
4
Peripheral Blood Lymphocyte and Monocyte Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral whole blood samples were collected in sterile BD Vacutainer® EDTA (ethylenediamine-tetraacetic acid) blood collection tubes (BD Biosciences). The samples were incubated in BD Trucount™ tubes (BD Biosciences) with the following monoclonal antibodies to identify lymphocyte sub-populations (BD Biosciences): anti-CD45-PerCP (clone 2D1), anti-CD3-FITC (clone UCHT-1), anti-CD4-APC (clone RPA-T4), anti-CD8-PE (clone RPA-T8), anti-CD16-PE (clone 3G8), anti-CD19-APC (clone HIB19), anti-CD56-PE (clone NCAM16.2). The following antibodies were used for monocyte sub-populations (BD Biosciences): anti-CD45-APC (clone 2D1), anti-HLA-DR-PerCP (clone L243), anti-CD14-FITC (clone M5E2), and anti-CD16-PE (clone 3G8). The samples were incubated at 4 °C for 30 min and then were treated with FACS Lysing Solution (BD Biosciences) until the erythrocytes were lysed and the cells were immediately processed in the FACSCanto flow cytometer (Becton–Dickinson Immunocytometry Systems, Palo Alto, CA) along with 10,000 beads per tube. The results were analyzed with FACSDiva Software (BD Biosciences) and the absolute numbers of lymphocytes and monocytes subsets were calculated on the basis of bead counts. Monocyte populations were classified as classical (CD14++ CD16−), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++).12 (link)
5
Isolation and Culture of Human Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Paque Plus; GE Healthcare, Chicago, IL, USA) of buffy coat preparations from anonymous donors (Institute of Transfusion Medicine, Ulm University). Monocytes were selected from PBMCs by adherence on plastic. Monocyte-derived macrophages (referred to as “macrophages”) were generated by treatment with GM-CSF (10 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) cell culture medium supplemented with L-glutamine (2 mM; PAN Biotech, Aidenbach, Germany), HEPES (10 mM; Biochrom, Berlin, Germany), Penicillin/Streptomycin (100 µg/mL; Biochrom, Berlin, Germany) and 5% heat-inactivated human serum (Lonza, Basel, Switzerland) for 6 d. After culture, macrophages were detached with EDTA (1 mM; Sigma-Aldrich, Steinheim, Germany). Phenotypic characterization by flow cytometry demonstrated that macrophages expressed CD68 (anti–CD68-FITC, clone Y1/82A; BD Biosciences, Franklin Lakes, NJ, USA) and MHCII (anti–HLA-DR-PerCP, clone L243; BD Biosciences, Franklin Lakes, NJ, USA) as described [53 (link)].
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!