The fluorochrome 4’, 6-Diamidine-2’-pheny-lindole dihydrochloride (DAPI), was used to detect DNA. From the 10 neuropathologically confirmed AD cases, and four control cases, 7 μm thick cortical sections were cut on a cryostat, postfixed with methanol for 2 min and stained with 3 μg/ml of DAPI (Boehringer, 236 276) in methanol for 15 min at 37C. The sections were rinsed in distilled water for 5 min and were mounted with gum arabic, coversliped and examined with a fluorescence microscope either in UV light, using G 365/11 excitation and LP 397 barrier filters, or using Bp 485/20 excitation and LP 520 barrier filters. Frozen sections of control cases where also stained with DAPI. In order to remove DNA, another set of sections before staining with DAPI was treated with 1 mg/ml of DNase I (Boehringer, 1284 932) diluted in PBS containing 5mmol/ml of Mg++, at pH 7.8 at 37°C for 3 h. The same procedure was also carried out using RNase free DNase I (Boehringer, 776 785). In order to eliminate the possibility of an unspecific binding of the DAPI to amyloid, a set of DNase I treated sections were post-stained with thioflavin S, widely used for the demonstration of amyloid in AD [32 ]. Smears of strains B31, ADB1 and ADB2 of B. burgdorferi were treated and examined in the same manner.
Dnase 1
Manufactured by Boehringer Ingelheim
Sourced in Germany
DNase I is a lab equipment product that functions as an endonuclease enzyme. It catalyzes the hydrolytic cleavage of DNA, specifically the phosphodiester bonds within the DNA molecule.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 2, by Worthington (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Collagenase, by Boehringer Ingelheim (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Streptomycin, by Merck Group (1 mentions)
25 cm2 culture flask, by Corning (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Boehringer Ingelheim (1 mentions)
Live dead dye, by Thermo Fisher Scientific (1 mentions)
Il 17, by Thermo Fisher Scientific (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Ack solution, by Lonza (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Collagenase d, by Roche (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Cell culture flask, by Corning (1 mentions)
8 protocols using dnase 1
1
DAPI Staining for Amyloid Detection in Alzheimer's Disease
2
Autologous Tumor Cell Vaccine Production
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Patients underwent a total nephrectomy or, if the kidney was already removed before inclusion, a metastasectomy. The tissue that was not used for pathologic diagnosis and staging was transferred to our vaccine production laboratory within 48 h of the surgery. The tumor tissue was then dissociated as previously described [13 (link), 24 (link)]. Briefly, tumor tissue was cut into small pieces and subsequently incubated in a 0.1% DNase I, 0.14% collagenase (Boehringer) solution. After 45 min incubation at 37 °C, single cells were harvested and remaining tumor fragments again suspended in a DNase/collagenase solution; this cycle was repeated 3–4 times, after which single cells were harvested through a 100 µm gauze, a sample for bacteriology control was taken and viability was tested using trypan blue exclusion. Cells were aliquoted (at 15–20 × 106 viable cells per vial) and cryopreserved using a linear freezer. Vials were stored in liquid nitrogen until vaccination. Prior to vaccination, the frozen tumor cells were irradiated (20,000 rad), thawed, counted and assessed for viability. For each patient, we aimed to produce as many vaccines as possible.
3
Isolation of Lung Cell Suspensions
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Lung resection specimens were processed as described previously to obtain single cell suspensions [14 (link)]. In brief, lung tissue was rinsed with physiological water (0.9% NaCl) to remove residual blood. The lung tissue was cut into fine pieces and digested for 45 minutes at 37°C in digestion medium (Roswell Park Memorial Institute medium (RPMI) 1640 supplemented with 5% fetal calf serum (FCS), 2 mM L-glutamine, 0.05 mM 2-mercaptomethanol (all Invitrogen), 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma-Aldrich), 1 mg/ml collagenase type 2 (Worthington Biochemical), and 0.02 mg/ml DNase I (grade II from bovine pancreas; Boehringer Ingelheim)). Cells were resuspended in 10 mM ethylenediaminetetraacetic acid (EDTA) for 5 minutes at room temperature on a shaker. Next, cells were filtered through a 40-μm cell strainer and mononuclear cells were isolated with Ficoll-PaqueTM plus (GE Healthcare). Finally, cells were subjected to red blood cell lysis (S1 Fig ).
4
Lung Cell Isolation and Preparation
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The pulmonary circulation was rinsed with saline/ethylenediaminetetraacetic acid to remove the intravascular pool of cells. The cell suspensions of the lungs were obtained using digestion medium (RPMI-1640 supplemented with 5% FSC, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol [Gibco, Invitrogen, Paisley, UK], 100 U/mL penicillin, 100 mg/mL streptomycin [Invitrogen], 1 mg/mL collagenase type 2 [Worthington Biochemical, Lakewood, NY, USA], and 0.02 mg/mL DNase I [grade II from bovine pancreas; Boehringer Ingelheim, Ingelheim, Germany]) for 45 minutes at 37°C and at 5% CO2. Red blood cells were lyzed by using ammonium chloride buffer. Cells were counted using a Bürker hemocytometer and resuspended (107 cells/mL) in phosphate buffered saline.
5
Isolation and Characterization of Intestinal Fibroblasts
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Human and murine intestinal fibroblasts were isolated and cultured as previously described [60 (link)]. In brief, either endoscopic biopsies or surgical specimens were taken from the designated area of the mucosa. Surgical specimens were cut into 1-mm pieces. Epithelial cells were removed by washing the mucosa in Hank’s Balanced Salt Solution ((HBSS) without Ca2+ and Mg2+) with 2 mM EDTA, followed by digestion of remaining tissue for 30 min at 37 °C with 1 mg/mL collagenase 1, 0.3 mg/mL DNase I (Boehringer, Mannheim, Germany), and 2 mg/mL hyaluronidase in PBS. Isolated cells were cultured in 25-cm2 culture flasks (Costar, Bodenheim, Germany) in DMEM containing 10% FCS, penicillin (100 IE/mL), streptomycin (100 μg/mL), ciprofloxacin (8 μg/mL), gentamycin (50 μg/mL), and amphotericin B (1 μg/mL). Non-adherent cells were removed by subsequent changes of medium. Cells were characterized by immunocytochemistry and used between passages 6 and 12.
6
Isolation and Analysis of Lymph Node and Spleen Cells
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Popliteal and inguinal LNs were isolated and a single-cell suspension was prepared by incubating LN with 1 mg/mL collagenase D (Roche) and 0.04 mg/mL DNase I (Boehringer) in 5% FSC containing DMEM, for 30 min at 37°C. Organ pieces were passed through a 70-μm cell strainer and stained for cell-specific markers. The following fluorochrome-labeled antibodies were used: CD11c, CD8a (all from eBioscience), F4/80, Live/Dead Aqua cell stain (all from Life Technologies).
Spleens were isolated smashed through a 70-μm cell strainer using the plunger of a syringe (Falcon), red blood cells were lysed with ACK solution (Lonza). The following fluorochrome-labeled antibodies were used: CD3, CD8a, Live-Dead dye, IFNγ, TNFα, and IL-17 (eBioscience).
Primary culture of DCs from LNs of a naïve mice were harvested and incubated for 24 h with 10 nM of E749-51 conjugated with Alexa488 and 1 μg/mL of Qβ conjugated with Alexa647. CD11c+ F4/80− DCs were subsequently analyzed by flow cytometry for uptake of peptide and Qβ.
Spleens were isolated smashed through a 70-μm cell strainer using the plunger of a syringe (Falcon), red blood cells were lysed with ACK solution (Lonza). The following fluorochrome-labeled antibodies were used: CD3, CD8a, Live-Dead dye, IFNγ, TNFα, and IL-17 (eBioscience).
Primary culture of DCs from LNs of a naïve mice were harvested and incubated for 24 h with 10 nM of E749-51 conjugated with Alexa488 and 1 μg/mL of Qβ conjugated with Alexa647. CD11c+ F4/80− DCs were subsequently analyzed by flow cytometry for uptake of peptide and Qβ.
7
Lung Cell Isolation and Dissociation
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The left lung lobe was collected, minced and incubated with digestion medium (RPMI 1640 supplemented with 5 % FCS, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol [Gibco, Invitrogen, Paisley, United Kingdom], 100 U/ml penicillin, 100 mg/ml streptomycin [Invitrogen], 1 mg/ml collagenase type 2 [Worthington Biochemical, Lakewood, NY], and 0.02 mg/ml DNase I [grade II from bovine pancreas, Boehringer Ingelheim, Ingelheim, Germany]) for 45 min at 37 °C and 5 % CO 2 . Red blood cells were lysed by using ammonium chloride potassium lysing buffer. Cells were counted using a Bürker hemocytometer and resuspended (10 7 cells/ml) in PBS.
8
Isolation and Culture of Colonic Lamina Propria Fibroblasts
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Human CLPF of healthy controls (n = 3-5) were isolated and cultured as described previously [1] . Mucosa from surgical specimens was cut into 1-mm pieces, whereas biopsy specimens were used directly for the isolation of CLPF. Epithelial cells were separated in Hank's balanced salt solution without Ca 2+ and Mg 2+ (PAA Laboratories GmbH, Linz, Austria) with 2 m M EDTA (Sigma, Deisenhofen, Germany). The remaining tissue was rinsed and digested for 30 min at 37 ° C with 1 mg/ml collagenase 1 (Sigma), 0 • 3 mg/ml DNase I (Boehringer Ingelheim, Ingelheim, Germany) and 2 mg/ml hyaluronidase (Sigma) in PBS (Gibco, Karlsruhe, Germany). Isolated cells were cultured in 25 cm 2 cell culture flasks (Costar, Bodenheim, Germany) with Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS, penicillin (PAA Laboratories), streptomycin 10 mg/ml (PAA Laboratories), ciprofloxacin 2 mg/ml (Bayer, Leverkusen, Germany), gentamycin 50 mg/ml (PAA Laboratories) and amphotericin B 1 mg/ml (Biochrom, Berlin, Germany). Non-adherent cells were removed by subsequent changes of medium. The remaining cell culture contained CLPF and was used between passages 3 and 8. The study was approved by the University of Regensburg Ethics Committee.
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