PBMCs were isolated from whole blood using Ficoll-Paque. Briefly, 6 mL of EDTA-anticoagulated blood was diluted with an equal volume of phosphate-buffered saline, pH 7.4 (PBS), containing 0.05 M ethylenediaminetetraacetic acid (EDTA; Invitrogen, Waltham, MA, USA). A total of 12 mL of diluted blood was layered over 24 mL of the Ficoll-Paque PLUS (GE Healthcare, Casoria, NA, Italy). Gradients were centrifuged at 400× g for 30 min at room temperature in a swinging-bucket rotor with no brake or acceleration. The cell interface was carefully removed by pipetting and washed with PBS-EDTA by centrifugation at 250× g for 10 min. PBMC pellets were suspended in ammonium chloride solution (Stemcell Technologies, Vancouver, BC, Canada) and incubated for 10 min at room temperature on a mixing platform in order to lyse contaminating red blood cells. Isolated PBMC were finally washed with PBS-EDTA and then cryopreserved in liquid nitrogen in fetal calf serum (FCS; Invitrogen, Waltham, MA, USA) containing 10% dimethyl sulfoxide (DMSO; Thermo Fisher Scientific, Waltham, MA, USA) and stored until required for downstream analyses.
Ammonium chloride solution
Manufactured by STEMCELL
Sourced in Canada, France
Ammonium chloride solution is a laboratory reagent used as a source of ammonium ions in various applications. It is a clear, colorless liquid with a pungent odor. The concentration of the solution may vary depending on the product specifications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Hyaluronidase, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Uv live dead fixable stain, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Cd11b, by BioLegend (3 mentions)
Lsrii cytometer, by BD (3 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Roche (2 mentions)
Lymphocyte separation medium, by Corning (2 mentions)
K2 edta tube, by BD (2 mentions)
Methocult gf m3434, by STEMCELL (2 mentions)
Streptavidin particles plus dm, by BD (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Dispase, by STEMCELL (2 mentions)
74 protocols using ammonium chloride solution
1
Isolation and Cryopreservation of PBMCs
2
Colorectal Tumor Spheroid Culture Protocol
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Primary colorectal tumor samples were obtained with patient informed consent, as approved by the Ottawa Health Science Network Research Ethics Board (OHSN-REB) via the Global Tissue Consenting initiative (GTC, OHRI), the University Health Network Research Ethics Board, and from Celprogen Inc. (#36112-39P, Torrance, CA). Tumor tissues were mechanically minced and incubated with Collagenase A (3 mg/mL) for 60 min at 37°C and filtered using a 40-μm cell strainer. Red blood cells were removed using ammonium chloride solution (STEMCELL Technologies) (5 min). Isolated cells were maintained as spheroids in ultra-low adhesion flasks with DMEM/F-12 (Gibco) (1:1 ratio) supplemented with penicillin-streptomycin (1%) (Gibco), L-glutamine (2 mM) (Gibco), nonessential amino acids (1X) (Gibco), sodium pyruvate (1 mM) (Gibco), HEPES (Gibco), heparin (4 μg mL−1), B27 supplement (GIBCO), N2 supplement (GIBCO), lipids mixture (Sigma), EGF (20 ng/mL) and bFGF (10 ng/mL) (Kreso and O'Brien, 2008 (link)).
3
Murine Myeloid Cell Immunophenotyping
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Peripheral blood was isolated from mice and RBCs were lysed with ammonium chloride solution (Stemcell Technologies). Cells were subsequently plated and blocked with anti-mouse FcR antibody (αCD16/CD32, BioLegend) in FACS buffer (PBS with 2% BSA and 1mM EDTA). Cells were then surface-stained with CD11b-BV421 (clone M1/70, BioLegend), F4/80-APC (clone BM8, eBioscience), Ly6G-FITC (clone 1A8, Miltenyi Biotec), CD115-PerCP-Cy5.5 (clone AFS98, Biolegend), and TLR2-PE-Vio770 (clone REA109, Miltenyi Biotec) according to manufacturer’s recommendations. Data were collected on the BD LSRII Flow Cytometer (BD Biosciences) platform and exported for analysis via FlowJo (v.9.0; Treestar, Inc, Ashland, OR).
4
Isolation and Stimulation of Splenic T Cells
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The extracted spleens were mashed with the plunger of 5-ml syringe onto a 100 μm cell strainer (BD Biosciences) with washing buffer (DMEM containing 2.5% FBS and 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The separated spleen cells were treated with an ammonium chloride solution (STEM CELL, British Columbia, Canada) for 10 min at 4 °C to lyse the red blood cells. The splenocytes were washed with washing buffer and were then filtered through a 40 μm cell strainer (BD Biosciences). Finally, the cells were incubated with RPMI 1640 containing 10% FBS, 50 μM 2-ME, 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and antibiotics (100 unit ml−1 penicillin and 100 μg ml−1 streptomycin) in a 24-well plate (TPP) coated with 1 μg ml ml−1 of anti-CD3 (eBioscience, San Diego, CA, USA) and 1 μg ml−1 of anti-CD28 (eBioscience) antibodies for 12 h to re-stimulate the splenic T cells.
5
Isolating Mouse Cardiac Cells Post-MI
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Blood was collected from the heart of Zsgreen mice after MI surgery. Blood was incubated on ice for 15 min with ammonium chloride solution (STEMCELL Technologies, 07800, Canada). After centrifugation, the supernatant was discarded and the cells were washed with RoboSep™ Buffer (STEMCELL Technologies, 20104, Canada) and resuspended. Subsequently, the sample was transferred to a polystyrene round-bottom tube with 25 μL normal rat serum (Abcam, ab13551, USA) and 25 μL selection cocktail (STEMCELL Technologies, 14766, Canada) in each well, and the sample was incubated at room temperature for 5 min. RapidSpheres™ (40 μL/1 mL) and RoboSep™ Buffer were added to the sample and the tube was placed in an EasySep™ magnet (STEMCELL Technologies, 18000, Canada) at room temperature for 3 min. After removing the supernatant from the tube in the magnet, the cells were resuspended in sterile PBS. The harvested cells were injected into BALB/c nude mice with or without MI.
6
Isolation of Bone-Derived Stromal Cells
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For preparation of BLCs, femurs and tibias were harvested, cleaned of excess muscle tissue, and placed into PBS/5% FCS. Bones were crushed in a mortar, and BM was removed for separate processing of central BM. The bones were fragmented with scissors and transferred to a 15-ml tube (Falcon). Bones were spun down, and excess PBS/FCS was removed. Cells were isolated by incubation at 37°C with type I collagenase (3 mg/ml; Worthington Biochemical Corporation) for 45 min with rotation in DMEM with 10% FCS. The cell suspension was collected by passing through a 70-µm cell strainer. Collagenase was then added for a further 45 min. Cells were collected and washed in PBS/5% FCS/2 mM EDTA to reduce clumping.
For analysis of central BM stromal cells, BM cells were incubated at 37°C with type IV collagenase (2 mg/ml; Worthington Biochemical Corporation) for 20 min with rotation in DMEM with 10% FCS, and RBCs were lysed with ammonium chloride solution (STEMCELL Technologies) at room temperature for 10 min. Subsequently, CD45+ cells were depleted with anti-CD45 MicroBeads (Miltenyi Biotec). Cells were collected and resuspended in PBS/5% FCS/2 mM EDTA for FACS analysis.
For analysis of central BM stromal cells, BM cells were incubated at 37°C with type IV collagenase (2 mg/ml; Worthington Biochemical Corporation) for 20 min with rotation in DMEM with 10% FCS, and RBCs were lysed with ammonium chloride solution (STEMCELL Technologies) at room temperature for 10 min. Subsequently, CD45+ cells were depleted with anti-CD45 MicroBeads (Miltenyi Biotec). Cells were collected and resuspended in PBS/5% FCS/2 mM EDTA for FACS analysis.
7
TNBC Breast Cancer FNA Acquisition
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Fine needle aspiration (FNA) samples were obtained from 21 g needle aspiration of a cT2N0 primary breast cancer, of BRCA2 −/− ER-, PR-, HER2- (TNBC) subtype. The FNA was expelled into DMEM media, then the needle was washed with DMEM media and the wash was also collected, repeating needle washing twice for a total of three washes. FNA samples were kept at 4°C until processing for DLP+. They were treated with 0.8% Ammonium Chloride Solution (StemCell) to lyse red blood cells prior to staining.
8
Isolation of Mammary Fat Pad Cells
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Mechanically dissociated inguinal, abdominal, and thoracic mammary fat pads were prepared into cell suspension for flow cytometry or fluorescence-activated cell sorting (FACS). The lymph node was removed from abdominal glands. Glands were chopped using a mechanical tissue chopper and digested for 1 h at 37 °C in digestion media [RPM1 containing 1%FBS, collagenase IA (Sigma, C9891), hyaluronidase (Sigma, H3506) and DNAse I (Worthington, LS002007)]. Tissue was washed with washing buffer (1X PBS containing 2% FBS) and centrifuged at 1,000 rpm for 5 min at 4 °C. Tissue was further digested using pre-warmed 0.25% Trypsin-EDTA (Thermo Fisher, 25200056), washed, and digested with 5 mg/ml of pre-warmed dispase II (Roche, 4942078001). Red blood cells were lysed using Ammonium Chloride Solution (Stem Cell Technologies, 07850). Cells were washed, resuspended and filter through a 70 µm cell strainer (Falcon, 08-771-2) and processed for downstream applications.
9
Isolation of Peripheral Blood Components
10
Visceral WAT Stromal Vascular Fraction Isolation
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Perigonadal visceral WAT was collected, the stromal vascular cell (SVC) fraction was separated, and the mature adipocyte fraction was enriched using a collagenase‐based approach, as described previously (Cho et al., 2014 (link)). Red blood cell (RBC) lysis was performed on SVC by incubating the cells in a 0.8% ammonium chloride solution (StemCell Technologies, Cambridge, MA), and the cells were subjected to staining for flow cytometry analysis. Cells were stained with the UV LIVE/DEAD fixable stain (Invitrogen) and then surface labeled for different combinations of the following markers: CD45, CD11b, CD11c, CD206, CD301, CD19, TCRβ, Ly6G, F4/80, and Ly6C (Biolegend, San Diego, CA) and fixed with 1% paraformaldehyde (Sigma Aldrich, St. Louis, MO). Samples were analyzed on an LSRII cytometer (BD Biosciences) or an Aurora cytometer (Cytek Biosciences). All flow cytometry data analysis was performed using FlowJo Software version 10.6.1 (BD Biosciences).
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