Murine retinal endothelial cells (mREC) were cultured at 80% confluency, as previously described [26 (link),36 (link)]. Spleens were removed, a single-cell suspension was generated, and incubated in erythrocyte lysis buffer (eBioscience) for 5 min at 37 °C, and then washed and counted. CD3+ T cells were negatively selected from isolated splenocytes using mouse CD3+ T cell enrichment columns (R&D). Cells were washed, and 1 × 105 T cells of non-diabetic or diabetic C57BL/6 or RORγt−/− mice were co-cultured with 3 × 105 murine retinal endothelial cells per well. Cells were stained with 10 μg/mL Propidium Iodide, incubated for 48 h, and imaged using a Leica DMI 600B inverted microscope. Additionally, co-cultured cells were then stained with 0.25 μg/tube CD144 (BD Bioscience) for flow cytometry quantification of cell viability, as previously described [36 (link)]. Alternatively, co-cultured cells were incubated with Annexin V (eBioscience) antibodies for apoptosis analysis per manufacturer’s directions.
Erythrocyte lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Erythrocyte lysis buffer is a solution used to selectively lyse or disrupt red blood cells (erythrocytes) in a sample. This process is commonly used in various analytical and research applications to isolate other cell types, such as leukocytes, for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Glucose, by Carl Roth (2 mentions)
Hepes, by Merck Group (2 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Mouse cd3 t cell enrichment column, by R&D Systems (1 mentions)
Dmi 600b inverted microscope, by Leica camera (1 mentions)
Cd144, by BD (1 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse il 17a, by R&D Systems (1 mentions)
40 μm cell strainer, by BD (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Fixation buffer, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 antibody, by Thermo Fisher Scientific (1 mentions)
C0130, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Collagenase, by Merck Group (1 mentions)
22 protocols using erythrocyte lysis buffer
1
Murine Retinal Endothelial Cell-T Cell Co-culture
2
Murine Corneal Fibroblasts and Bone Marrow-Derived Macrophages: In Vitro Stimulation
3
Isolation and Characterization of Human Neutrophils
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Neutrophils, or PMNs, were isolated from whole blood anti-coagulated with sodium citrate. Briefly, approximately 2 parts of blood were layered on 1 part of Polymorphprep before centrifugation at 500x g, 23 °C for 30 min (without brakes). The layer containing PMNs was harvested before erythrocyte lysis with Erythrocyte Lysis Buffer (eBioscience, USA). PMNs were then re-suspended in RPMI without phenol red supplemented with 10% foetal bovine serum (FBS). Flow cytometry was then performed on either LSR-II or LSR-Fortessa-X20 (Becton-Dickinson, USA) and the analyses were performed using Flowjo (Treestar, USA). PMNs were defined as CD45+CD66b+ cells and percentage of CD66b+ cells over live CD45+ was determined to be >95%, unless otherwise stated, before the start of experiments.
4
Isolation of Brain and Spleen Immune Cells
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Isolation of immune cells from brain was performed as previously described [52 (link), 54 (link)]. Briefly, brain hemispheres were mechanically homogenized in dissection buffer (HBSS, Gibco™, Germany), supplemented with 50 mM glucose (Roth, Germany) and 13 mM HEPES (pH 7.3, Sigma), using a glass potter, and then filtered through a 70-μm cell strainer (Falcon®, Corning, Germany). The cell suspension was washed (400 g, 10 min, 4 °C) with PBS and fractionated on a discontinuous 30%–70% isotonic Percoll® gradient (GE Healthcare, Germany) (800 g, 30 min without brake, 4 °C). After the removal of myelin debris, cells in the interphase comprising mononuclear cells were isolated and washed with FACS buffer (PBS w/o Ca2+/Mg2+, 2% v/v fetal bovine serum (FBS), 10 mM HEPES, 0.1% sodium azide), centrifuged (400 g, 10 min, 4 °C) and immediately used for flow cytometric analysis. To isolate immune cells from spleens, organs were homogenized and sieved through a 40-μm cell strainer (Falcon®, Corning). Isolated cells from spleen and blood samples were treated with erythrocyte lysis buffer (eBioscience, Germany), and washed twice with FACS buffer (400 g, 10 min, 4 °C) before staining and flow cytometric analysis.
5
Flow Cytometric Analysis of Corneal Leukocytes
6
Isolation and Sorting of Adipocytes and Stromal Vascular Cells
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Epididymal fat pads were excised from male C57BL/6 mice fed HFD, weighed, and rinsed three times in PBS containing low endotoxin BSA and EDTA (1 mm). Tissue suspensions were subjected to centrifugation at 500g for 5 min and then collagenase treated (1 mg/mL) (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C with shaking. Cell suspensions were passed through a 100-μm filter and centrifuged at 500g for 5 min. Adipocyte fractions were collected into QIAzol (Qiagen, Valencia, CA) for RNA extraction. Stromal vascular cell fraction pellets were then incubated with erythrocyte lysis buffer (eBioscience, San Diego, CA) for 5 min before centrifugation (300g for 5 min) and resuspended in fluorescence-activated cell sorting buffer. Stromal vascular cell fractions were incubated with Fc Block (BD Biosciences, San Jose, CA) for 20 min at 4°C before staining with fluorescently labeled primary antibodies. The antibodies used were as follows: anti-mouse CD45 Alexa Fluor 700 and anti-mouse F4/80 antigen PerCP-Cyanine5.5. Cells were sorted using a MoFlo sorter (Beckman Coulter, Brea, CA) into RTL buffer (Qiagen).
7
SAH Patient Peripheral Blood Profiling
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The peripheral blood of the SAH patients and controls was retrieved in EDTA blood tubes (S-Monovette® Sarstedt, Germany). Erythrocyte lysis was achieved with erythrocyte lysis buffer (eBioscience, Germany) at room temperature. Afterwards, centrifugation of the cells was carried out at 350×g for 5 min at 4 °C. A volume of 2 mL of an ice cold FACS flow cytometry buffer (BD Biosciences, Germany) was used to wash the cells after discarding the supernatant. Cells were resuspended in 1 mL of FACS buffer after washing and then, were counted using countess cell counting slides (Catalog # C10283, Eugene, Oregon, USA) by using Countess™ automated cell counter (ThermoFischer scientific, Germany). The final concentration of cells was adjusted to one million cells per 100 µL with the FACS buffer. Next, flow cytometry tubes (5 mL; Catalog # 55.1578, Sarstedt, Germany) were labelled for stained cells and fluorescence minus one (FMO) controls and 100 µL aliquots of the cells were dispensed into the respective tubes. A drop of Ultracomp eBeads (eBioscience, Germany) was dispensed into each tube containing 100 µL of FCS buffer for the acquisition of the single stained compensation controls. Next, Human Fc block pure (Catalog # 564220, BD Biosciences, Germany) was added to the cells and the cells were further incubated for 10 min on ice30 .
8
Isolation of Splenocytes from Immunized Mice
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Splenocytes from Ag immunized mice, with or without tumor inoculation, were isolated by pressing the spleen through a 100 μm cell strainer (Becton Dickinson). After treatment with an erythrocyte lysis buffer (eBioscience), the remaining cells were washed with PBS and resuspended in either 1% BSA/PBS for splenocyte proliferation assay or RPMI-1640 containing 10% FBS for Treg cell isolation.
9
Spleen Cell Cytokine Profiling via ELISA
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On day 42, the spleen tissues of BALB/c were aseptically isolated. The spleen of each animal was crushed through a 200‐mesh cell strainer in RPMI 1640 medium and centrifuged at 300 g for 5 min. Afterwards, the contaminating red blood cells were lysed using erythrocyte lysis buffer (0.9% ammonium chloride, eBioscience) for 5 min at room temperature. The resulting cell pellet was washed three times with complete RPMI 1640 medium. Subsequently, the splenocytes (2.5 × 106) were co‐incubated with the indicated antigens at 37°C with 5% CO2 and 95% humidity (5 µg of polysaccharide antigen in PBS or PBS alone). Cells were stimulated with concanavalin A as a positive control. After 72 h of incubation, culture supernatants were collected, and the concentrations of IFN‐γ, IL‐2 and IL‐4 were determined using commercially available ELISA kits (Dakewe) according to the manufacturer’s instructions.
10
Phagocytosis Assay for CD31 Knockdown PBM
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