Red blood cell lysis solution

Human umbilical cord vein blood was obtained from the MD Anderson Cancer Center Cord Blood Bank. Blood was diluted 1:2 in sterile PBS and separated using Leucosep tubes (Grenier Bio-One) and Lymphocyte Separation Media (Corning) according to the manufactures’ instructions. Following separation, the lymphocyte layer was treated with red blood cell lysis solution (eBioscience) and washed in PBS/FBS. CD34⁺ stem cells were isolated using the EasySep Human CD34 Positive Selection Kit (Stem Cell Technologies) according to the manufacturer’s instructions.
Isolated CD34⁺ cells were plated in 24 well plates at 40,000 cells/mL in IMDM media (Gibco) containing 20% BIT Serum substitute (Stem Cell Technologies), 20μg/mL Human LDL (Stem Cell Technologies), 100μM 5x10^-2M β-Mercaptoethanol (Invitrogen), and 1× StemSpan Megakaryocyte Expansion Supplement. Cells were incubated at 37°C at 5% CO₂ in a humidified incubator. Four days post isolation, cells were split 1:2. Seven days post isolation, cells were replated at 3 to 5x10⁵ cells/ml. Differentiation was assessed on days 4, 7, 11, and 14 post isolation using flow cytometry [30 (link)].

Hu-NSG mice were infected with 10⁶ plaque forming units of virus delivered via subcutaneous injection. Clinical signs of infection were assessed on days 2, 4, 6, 8, 10, 12, and 14 post infection. Mice were anesthetized via isoflurane inhalation, and temperature and erythema were measured via rectal thermometer and DSMII Colormeter (Cortex Technologies), respectively. Approximately 20μL of blood were obtained via retroorbital bleed to quantify viremia. Blood was transferred to microcentrifuge tubes and allowed to clot before centrifuging at 500g for 15 minutes to separate out serum. Trizol LS (Ambion) was added to serum samples according to manufacturer’s instructions, and samples were stored at -70°C until RNA extractions and qRT-PCR were performed. On day 10 post infection, blood was collected via retroorbital bleed to perform platelet counts.
On days 8 and 14 post infection, mice were euthanized via isoflurane overdose. Femurs were dissected out of euthanized mice, and bone marrow was flushed out of femurs using a 5/8 inch 25 gauge needle and sterile PBS with 2% FBS. Marrow was forced through a 0.45μM filter to create a single cell suspension. Cells were treated with red blood cell lysis solution (eBioscience) and washed with sterile PBS/FBS. Infected megakaryocytes were identified using flow cytometry.

A proliferation assay was performed to evaluate cell-mediated immunity. Twenty-one days post primary immunization, blood and spleens were harvested. Lymphocytes in the blood were harvested using the gentle swirl technique [22 (link)] and plated in quadruplicate, in a 96-well plate at 10⁵ cells/well in RPMI-1640 without phenol red. Spleens were placed through a 70 μm cell strainer to obtain single cell suspensions. Red blood cells were lysed with Red Blood Cell Lysis solution (eBioscience). Splenocytes were then washed, suspended in RPMI and plated at 10⁶ cells/well. Each set of cells was incubated at 37°C, 5% CO₂ for 72 h with or without 4 μg/ml of either His-Fba, S. Typhimurium LPS, His-NetB, His-PlcC, or 1 μg/ml PMA. Cell proliferation was measured using the Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes). Mean absorbance value of antigen stimulated wells divided by mean absorbance of non-stimulated control wells was used to calculate stimulation index.

Leukocytes were purified from blood using red blood cell lysis solution (eBioscience, San Diego CA) following manufacturer's protocol. Peritoneal cells or leukocytes were labeled with antibodies for anti-mouse CD3 (eBioscience), CD4 (eBioscience), CD8 (eBioscience), B220 (BD Biosciences, San Jose CA), F4/80 (eBioscience), TLR2 (eBioscience), TLR4 (R&D Systems, Minneapolis MN), or RAGE (R&D Systems) following manufacturer's protocol. THP-1 and Jurkat, Clone E6-1 cells were grown in complete RPMI medium at a density of 2×10∧5 cells/mL, supplemented with 1 µM, 10 µM, 20 µM, or 30 µM curcumin, and collected after 10 min or 24 h. Data were collected on the BD FACS Calibur platform (BD Biosciences) using CellQuest Pro (v5.1, BD Biosciences) and exported for analysis via FlowJo (v.7.6.5; Tree Star, Inc, Ashland, OR).

Source leukocytes were obtained from the Gulf Coast Regional Blood Center (Houston, TX). PBMCs were isolated via density centrifugation using Leucosep centrifuge tubes (Grenier Bio-one) and lymphocyte separation media (Corning) according to manufacturers’ protocols. To remove any residual red blood cell contamination, PBMC pellets were then treated with red blood cell lysis solution (eBiosciences) according to manufacturer’s instructions. PBMCs were resuspended in RPMI 1640 media with 10% fetal bovine serum (FBS) and were plated at 1x10⁶ cells/well in a 24 well plate. PBMCs were stimulated with mosquito saliva (2μg salivary proteins per well), or the equivalent of 4 mosquito bites [37 ], to correspond with the number of infected mosquito bites required to produce dengue fever in humanized mice [7 (link)], and lipopolysaccharide (1mg/L media; Sigma), pokeweed mitogen (5mg/L media; Sigma), or untreated media. Supernatant samples were collected daily for five days post stimulation to assess cytokine production. On day five post stimulation, all PBMCs were assessed via flow cytometry.