The compound library, InhiTinib, and its related analogs were provided by the IRIC HTS platform. Because the analog ligands are proprietary, their structures are not shown here. CD3-CD28 dynabeads, CellTraceTM, Hoechst 33342, nerve growth factor (NGF), and MitoSoxTM Red were purchased from Thermo Fisher. Human and murine interferon (IFN)-gamma Quantikines were purchased from R&D System. Annexin-V, propidium iodide (PI) and anti-TUBB3 antibody were purchased from Biolegend. Lipopolysaccharide, glial-derived neurotrophic factor (GDNF), cytosine arabinoside (AraC), and the immunosuppressive agent Cyclosporin A were purchased from Sigma. Collagenase A and dispase II were purchased from Roche Applied Sciences. T-cell isolation kit and lymphoprep® were purchased from StemCell Technologies. Z-VAD-FMK pan-caspase inhibitor was purchased from APExBIO. Western blot antibodies against human and murine poly(ADP-Ribose) polymerase-1 (PARP-1) (Cat. #sc-8007 and 46D11) were purchased from Santa Cruz and Cell Signaling, respectively; γH2AX (Cat. #05-636) from EMD Millipore; and H4 and tubulin (Cat. #ab6161) from Abcam SPHEROTM. AccuCount Particles were purchased from Spherotech, Inc.
Accucount particles
Manufactured by Spherotech
Sourced in Germany
The AccuCount Particles is a laboratory instrument designed for precise particle counting and sizing. It utilizes advanced optical detection technology to accurately measure the size and concentration of particles in a sample. The core function of the AccuCount Particles is to provide reliable and consistent data on the characteristics of the particles under analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fc block, by BD (2 mentions)
Efluor 450, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 protein labeling kit, by Thermo Fisher Scientific (2 mentions)
Cd8 percp cy5, by BD (2 mentions)
Ly6c percp cy5, by BD (2 mentions)
Cd3 apc 145 2c11, by BD (2 mentions)
Cd4 apc, by R&D Systems (2 mentions)
Ccr2 apc, by R&D Systems (2 mentions)
Cd11c v450 or pe cy7 hl3, by BD (2 mentions)
Siglecf bv421 e50 2440, by BD (2 mentions)
Cd31 percp efluor 710 390, by R&D Systems (2 mentions)
Efluor 450, by R&D Systems (2 mentions)
Fasl apc, by R&D Systems (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Dispase 2, by Roche (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Collagenase a, by Roche (1 mentions)
7 protocols using accucount particles
1
High-throughput Screening of Inhibitor Analogs
2
Quantifying Leukocyte Subsets by Flow Cytometry
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Blood was collected in heparinized tubes and before centrifugation, 10 μl of blood sample were taken for determining the absolute number of leukocytes using CD45-FITC antibody (BioLegend, Fell, Germany) and AccuCount particles (Spherotech Inc., Lake Forest, IL, USA). After centrifugation plasma was taken and the cell pellet was depleted of erythrocytes by two treatment steps with 50 ml erythrocyte lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA pH 7.3). After washing with PBS, cells were incubated for 10 min on ice with FACS buffer (1 % fetal bovine serum in PBS) containing 1 μg of purified anti-mouse CD16/CD32 Fc block (eBioscience, Frankfurt am Main, Germany) per 106 cells. Cells were subsequently stained for 30 min at 4 °C in the dark with CD45-APC-Cy7, GR1-BV510 and Ly6C-PerCP-Cy5.5 (all from BioLegend) and then fixed for 10 min at room temperature with 1 % paraformaldehyde (PFA; Merck, Darmstadt, Germany) in PBS. Samples were analyzed using FACS Canto II flow cytometer and FACS Diva software (BD Bioscience, Heidelberg, Germany).
3
Multiparametric Flow Cytometry Analysis
4
Multicolor Flow Cytometry Analysis
5
In Vitro Differentiation of Human B Cells into Plasma Cells
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B cells were cultured in 96-well round-bottom plates (cat# 353072; Corning) at a density of 0.5 to 1.0 × 105 cells per well in a final volume of 150 μL of complete B-cell medium as previously described [34 (link)] Cells were stimulated with a combination of recombinant IL-21 (30 ng/mL), anti-CD40 (0.1 μg/mL), and anti-IgM F(abʹ)2 (5.0 μg/mL). IL-21 antibody was added at the initiation of the culture period at a final concentration of 0.003 to 10 μg/mL. B cells were cultured for 7 days. After 7 days of stimulation, B cells were stained for 30 min at 4°C with an antibody cocktail specific for PCs. PCs were labeled with PerCP Cy5.5–conjugated anti-human CD19 (cat# 340951; BD Biosciences), activated protein C (APC)–conjugated anti-human CD38 (cat# 340439; BD Biosciences), and PE-conjugated anti-human IgD (cat# 555779; BD Biosciences). Samples were washed, and AccuCount Particles (cat# ACBP-100-10; Spherotech, Lake Forest, IL) were added to all wells for enumeration of PCs. Cells were run on an LSR II flow cytometer (BD Biosciences) using FACSDiva software, and samples were analyzed with FlowJo software (version 9).
6
Multicolor Flow Cytometry Analysis
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Cells were suspended in 3% FBS 2mM EDTA in PBS and staining was performed in the presence of 2% NRS, 2% Fc block (BD), and fixable viability dye eFluor 450 (eBioscience). Cells were stained with the following antibodies from BD: CD3-APC (145-2C11), CD19- APC (1D3), CD11c-V450 or PE-Cy7 (HL3), NK1.1-APC (PK136), Ly6G-V450 (1A8), Ly6C-PerCP-Cy5.5 (AL-21), CD11b-PE (M1/70), B220-APC (RA3-6B2), CD45-APC (30-F11), SiglecF-BV421 (E50-2440), CD8-PerCP-Cy5.5 (53-6.7). And the following antibodies from eBioscience: CD103-APC (2E7), EpCAM-PE-Cy7 (G8.8), CD31-PerCP-eFluor 710 (390), MHC Class II (I-A/I-E) eFluor 450 (M5/114.15.2), FasL-APC (MFL3), CD4-APC (GK1.5), and from R&D: CCR2-APC (475301). Alexa Fluor 647 protein labeling kit (Thermo) was used to label the influenza M2 (E10) (Bourmakina and García-Sastre, 2005 (link)) antibody. Cells were fixed with 2% formaldehyde after staining and analyzed on an LSRII after gating for FSC/SSC, singlets, and live cells. Cells were quantitated by flow cytometry with AccuCount Particles (Spherotech). Sorting was performed similarly on FACSAria.
7
Murine Model of Papain-Induced Lung Inflammation
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Mice were anesthetized using isoflurane inhalation and challenged with 35 µg papain (Calbiochem) in 40 µl PBS administered to the lungs by oropharyngeal aspiration daily for three consecutive days. Samples were harvested on day 4. BAL was performed by three consecutive washes of 1 ml complete medium. The number of cells in BAL and lung samples was quantified by FACS analysis with AccuCount Particles (Spherotech). For lung pathology, mice were challenged with 35 µg papain in 40 µl PBS on days 1, 3, and 5, and samples were harvested on day 6. A blinded pathologist scored inflammation in lungs on day 6 using routine hematoxylin and eosin–stained paraffin sections. Both the right and left lungs were examined. Perivascular, peribronchiolar, and interstitial/alveolar inflammation was scored for percentage involvement (0% = 0, 1–25% = 1, 26–50% = 2, 51–75% = 3, and 76–100% = 4). Perivascular and peribronchiolar inflammation was also scored for the mean thickness of the inflammatory cuff (0 cells = 0, 1–3 cells = 1, and ≥4 cells = 2). The values were summed for each sample to determine the inflammation score. One mouse was excluded because of histological evidence of acute bronchopneumonia, and two mice were excluded for a paucity of bronchioles available for evaluation.
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