Routine CSF parameters CSF was drawn as part of routine clinical work-up. Peripheral blood was taken immediately after lumbar puncture. CSF cells were counted manually in a Fuchs Rosenthal chamber. CSF glucose and lactate levels were measured by an enzymatic-amperometric method using chip-sensor technology (EKF Biosen C-Line). Albumin CSF/serum ratios and intrathecal immunoglobulin synthesis were determined by nephelometry (Siemens ProSpec®). Fluorescence-associated cell sorting (FACS) of CSF immune cells was performed as described previously. 18 The following antibodies were used for staining: cluster of differentiation (CD) 45, V450; CD3, APC-Cy7; CD4, PerCP (all BD Bioscience); CD8, PE-Cy7; CD14, FITC; CD19, ECD; CD138, PE; and CD56, APC (all Beckman Coulter). We stained CD4+ T cells (CD45 + CD3 + CD4 +), CD8 + T cells (CD45 + CD3 + CD8+), monocytes (CD45+CD14+), natural killer (NK) cells (CD45+CD56+), B cells (CD45+CD19+CD138-) and plasmablasts (CD45+CD19+CD138+). The percentage of each subpopulation was determined in relation to all CD45 positive single cells using FlowJo (version 10.1). In the discovery cohort, B cells and plasmablasts staining was missing in seven patients and monocytes staining was missing in two patients. CSF parameters are summarized in Table 2.
Cd138 pe
Manufactured by Beckman Coulter
Sourced in United States
The CD138-PE is a lab equipment product manufactured by Beckman Coulter. It is a fluorochrome-conjugated antibody that binds to the CD138 antigen, also known as syndecan-1. CD138 is expressed on the surface of plasma cells and is a useful marker for the identification and quantification of plasma cells in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd14 fitc, by Beckman Coulter (6 mentions)
Cd56 pc7, by Beckman Coulter (5 mentions)
Cd45 krome orange, by Beckman Coulter (5 mentions)
Cd8 pacificblue, by Beckman Coulter (4 mentions)
Hla dr ecd, by Beckman Coulter (4 mentions)
Cd3 pc5, by Beckman Coulter (4 mentions)
Cd4 apc, by Beckman Coulter (4 mentions)
Cd19 apcalexafluor700, by Beckman Coulter (4 mentions)
Cd16 apcalexafluor750, by Beckman Coulter (4 mentions)
Cd56 apc, by Beckman Coulter (3 mentions)
Versalyse buffer, by Beckman Coulter (3 mentions)
Navios flow cytometer, by Beckman Coulter (3 mentions)
Cd19 ecd, by Beckman Coulter (2 mentions)
Cd4 percp, by BD (2 mentions)
Cd3 apc cy7, by BD (2 mentions)
Cd8 pe cy7, by Beckman Coulter (1 mentions)
Cd11b pe, by Beckman Coulter (1 mentions)
Cd15 pe cy5, by Beckman Coulter (1 mentions)
Cd45 pe cy7, by Beckman Coulter (1 mentions)
Fluo 4 am, by Beckman Coulter (1 mentions)
10 protocols using cd138 pe
1
Immunophenotyping of CSF Immune Cells
2
Multicolor Flow Cytometry Analysis
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The samples were separated into RBCs and WBCs. The RBC labeling antibodies were CD45-PE/Cy7 (IM3548, Beckman Coulter, USA), CD36-PE/Cy5 (Customized from BD Biosciences, USA), CD138-PE (A40316, Beckman Coulter, USA), and Fluo-4 AM (S1060, Beyotime, Shanghai, China), and the WBC labeling antibodies were CD45-PE/Cy 7, CD15-PE/Cy5 (B49217, Beckman Coulter, USA), CD11b-PE (IM2581U, Beckman Coulter, USA), and Fluo-4 AM. A Beckman Coulter FC500 flow cytometer was used to excite the fluorescence and collect the signal.
3
SRRM2 Expression in Tumor Cells
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Heparin-anticoagulated bone marrow and peripheral blood specimens were collected, and two tubes were labeled in parallel for each specimen. Adjust the number of nucleated cells in each tube to 5–8 × 105. Add mouse anti-human SRRM2-specific recombinant antibody and matching isotype control antibody to two parallel tubes and incubate with all cells in the sample. After incubation at room temperature for 30 min, add 1.5 ml of saline and wash 3 times, centrifuge at 300 g/5 min and resuspend in 200 μL saline. Goat anti-rat IgG polyclonal to FITC fluorescent antibody (Abcam, batch ab150165, Cambridge, UK) and other fluorescent-labeled monoclonal antibodies (CD138-PE, CD38-APC,CD45-PC7, Beckman Coulter, Miami, FL, USA) were added, incubated for 15 min, and then treated with NH4Cl for 10 min for flow cytometry analysis. For the detection of all clinical samples, we uniformly used a flow cytometry protocol with four-color fluorescent labeling (FITC, PE, APC, and PC7) to analyze the expression of SRRM2 in tumor cells and other normal blood cells. Data acquisition and analysis were performed using a flow cytometer (Cytoflex S, Beckman Coulter) and CytExpert software (Beckman Coulter). A minimum of 105 nucleated cells and 500 target cells were obtained for most samples. For samples with fewer target cells, we obtained more cells by centrifugation or extended collection time.
4
Immunophenotyping of Peripheral Blood and CSF Cells
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Single-cell suspensions from human peripheral blood mononuclear cells and CSF cells were stained for 30 min at 4 °C with the appropriate combination of indicated fluorescence-labeled monoclonal antibodies in PBS, containing 0.1% sodium azide and 0.1% bovine serum albumin (BSA) following treatment with VersaLyse (Beckman Coulter GmbH, Krefeld, Germany) according to the manufacturer’s instructions. The following monoclonal antibodies were used at 1:200 dilutions: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). Flow cytometric analysis of stained cells was performed following standard protocols. Cells were analyzed on a Navios™ flow cytometer (Beckman Coulter) using Kaluza Analysis Software (V2.1, Beckman Coulter) and presented using Prism 6.0 (Graph Pad).
5
Flow Cytometric Immune Cell Profiling
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CSF and EDTA blood samples were obtained from all patients and processed as described previously (19 (link)). Briefly, cells from CSF or 100 μl EDTA blood were incubated in VersaLyse buffer (Beckman Coulter; Brea, CA) for 10 min and subsequently washed three times with PBS supplemented with 2% heat-inactivated FCS and 2 mM EDTA. Following incubation with fluorochrome-conjugated antibodies (CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange, all Beckman Coulter), cells were acquired on a Navios flow cytometer (Beckman Coulter). Analysis was conducted with Kaluza V1.2. Lymphocytes, monocytes, and granulocytes were selected based on forwards scatter channel, sideward scatter channel, CD14, and CD45 expression characteristics. Lymphocytes subsets were selected as CD3+CD4+ (T helper cells), CD3+CD8+ (cytotoxic T cells), CD3+HLA-DR+ (activated T cells), CD3−CD56+ (NK cells), CD3−CD19+ (B cells), CD3−CD19+CD138+ (plasma cells), whereas monocyte subsets were selected as CD14+CD16− (classical monocytes), CD14+CD16+ (non-classical monocytes) cells.
6
Flow Cytometric Analysis of Immune Cells in CSF
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Flow cytometric analysis of immune cell subsets was performed as described previously [20 ]. Shortly, fresh CSF was immediately spun down (300 g for 10 min), the supernatant removed, and the pellet resuspended in phosphate-buffered saline (PBS) (PAA, Pasching, Austria) with 2% fetal calf serum (FCS) (Invitrogen, Darmstadt, Germany). After incubating with our antibody mix (20 min at 4 °C), cells were spun down, washed, and resuspended in PBS wash solution (including 2% FCS) for flow cytometric analysis (Beckman Coulter Cyan, Brea, CA, USA). The following antibodies were used for staining: CD4 PerCP, CD3 APC-Cy7, CD45 VM (all BD Bioscience, Bedford, MA, USA), CD19 ECD, CD56 APC, CD14 FITC, and CD138 PE (all Beckman Coulter). This allowed differentiating CD4 T cells (CD45+CD3+CD4+), CD8 T cells (CD45+CD3+CD8+), monocytes (CD45+CD14+), NK cells (CD45+CD56+), B cells (CD45+CD19+CD138−), and plasmablasts (CD45 CD19+CD138+).
7
Multiparameter Flow Cytometry for Immune Cells
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Multiparameter flow cytometry of immune cells in PB and CSF samples was done as described previously (24 (link), 29 (link)). During lumbar puncture CSF was sampled into polypropylene tubes. All CSF samples were processed in < 20 min. Cells were isolated from CSF by centrifugation (15 min, 290 g, 4°C) and subsequent incubation in VersaLyse buffer (Beckman Coulter, Germany). PB samples were collected in EDTA monovettes and cells were isolated by using VersaLyse buffer. For immunostainings, the following fluorochrome-conjugated antibodies were used: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APC-Alexafluor700, CD16-APC-Alexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all from Beckman-Coulter). Data acquisition was performed with a Navios flow cytometer (Beckman-Coulter). Gating strategy for Leukocytes and Monocytes is described and illustrated in Supplementary Figure 1 .
8
Multiparametric Flow Cytometry of CSF and Blood
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CSF samples were collected in polypropylene tubes and were processed within 20 min. Cells were obtained from EDTA blood by erythrocyte lysis using VersaLyse buffer (Beckman Coulter, Krefeld, Germany) following the manufacturer's instructions. Cells were obtained from CSF by centrifugation (15 min, 290g, 4°C) and incubation in VersaLyse buffer. Cells were stained for 30 min at 4°C using the following fluorochrome-conjugated antibodies: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). T-cell subpopulations were further analyzed using the following fluorochrome-conjugated antibodies: CD45RA-FITC, CD27-PE, CD3-ECD, CCR7-PC5.5, CD25-PC7, CD56-APC, CD127-APCAlexafluor700, CD62L-APCAlexafluor750 or PD1-APCAlexafluor750, CD8-PacificBlue, and CD4-KromeOrange (obtained from Beckman Coulter or Ebioscience, Frankfurt, Germany). After washing, all samples were analyzed using the Navios™ flow cytometer (Beckman Coulter, Germany). The gating strategy to determine HLA-DR expression on CD4+ and CD8+ T cells and CD4+ and CD8+ T-cell subpopulations are described in Figures S1 and S2.
9
Daratumumab-Mediated Cytotoxicity in Multiple Myeloma
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BM-MNCs were obtained from 47 daratumumab-naïve MM patients (17 newly diagnosed MM and 30 RRMM patients). These patients were not treated in the DARA-ATRA study. Samples contained 2.0%–67% CD138+ tumor cells, as well as autologous effector cells and immune suppressive cells. The frequencies of MM cells, NK cells, and CD16+ NK cells in these samples at the start of the assay were determined by staining 1.0 × 106 cells with HuMax-003 FITC (Genmab/Janssen), CD138 PE, CD45 Krome Orange, CD56 PC7 (all Beckman Coulter), CD3 V450, and CD16 APC (both BD Biosciences). Sample viability at start of the assays, assessed using 7-AAD (BD Biosciences) was >95%. BM-MNCs were incubated with daratumumab 10 µg/mL in duplicate for 48 hours, after which MM cell-specific lysis was assessed by flow cytometric analysis, as described above.
10
Evaluating Apoptosis and CD138+ Cells in Multiple Myeloma
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ARP‐1 and H929 cells, seeded at a density of 2 × 105 cells/well in 6‐well plates, were treated with/without SM (5 μM) for 24 h. Apoptotic cells were quantified using a fluorescence‐activated cell sorter (FACS) Vantage SE flow cytometer (Beckman, CA, USA) after staining with Annexin V‐FITC/PI staining kit (Vazyme Biotech Co., Ltd, Nanjing, China).
Primary BM‐MNCs, isolated from the bone marrow of newly diagnosed MM patients, were treated with/without SM (5 μM) and/or BTZ (5 nM) for 24 h. After incubation with conjugated antibody CD138‐PE (Beckman Coulter Immunotech, Marseille, France) for 15 min, the percentage of CD138+ cells was analysed under a FACS Vantage SE flow cytometer (Beckman Coulter, Fullerton, CA, USA).
Primary BM‐MNCs, isolated from the bone marrow of newly diagnosed MM patients, were treated with/without SM (5 μM) and/or BTZ (5 nM) for 24 h. After incubation with conjugated antibody CD138‐PE (Beckman Coulter Immunotech, Marseille, France) for 15 min, the percentage of CD138+ cells was analysed under a FACS Vantage SE flow cytometer (Beckman Coulter, Fullerton, CA, USA).
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