Lung tissue was transported in Hank’s balanced salt solution (HBSS, Life Technologies) on ice immediately after surgery. Half of the tissue was embedded, and the rest was cut into 1-mm3 pieces and digested with collagenase I (2 mg/mL) and IV; (1 mg/mL) in a 15 mL conical tube (BD Falcon) at 37°C for 30 min on a tube revolver (Thermo) with frequent agitation. All samples were then filtered through 70 μm and 40 μm nylon mesh filters (BD Biosciences) and centrifuged at 4°C at 400 x g for 5 min. The cell pellet was suspended in red blood cell lysis buffer, centrifuged and resuspended in PBS with 0.04% FBS. Following dissociation, single-cell suspensions were stained with 7-aminoactinomycin D (7-AAD) in a dark room for 15 min before being analyzed by flow cytometry for live-cell sorting with a MoFloAstrios EQ (Beckman Coulter). Cell suspensions were added to the Master Mix to achieve a final number of 8000 cells per reaction for scRNA-seq.
Nylon mesh filter
Manufactured by BD
Sourced in United States, United Kingdom
Nylon mesh filters are laboratory equipment designed to separate solid particles from liquids or gases. They feature a woven nylon material that allows the passage of the fluid while retaining particulates of a specific size range. These filters are used for filtration and purification purposes in various scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Acrodisc syringe filter, by Pall Corporation (3 mentions)
Supor membrane, by Pall Corporation (3 mentions)
Collagenase 4, by Merck Group (2 mentions)
15 ml conical tube, by BD (1 mentions)
Tube revolver, by Thermo Fisher Scientific (1 mentions)
Moflo astrios eq, by Beckman Coulter (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Glucose dulbecco s modified eagles medium dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Brefeldin a, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
14 protocols using nylon mesh filter
1
Lung tissue dissociation and single-cell sorting
2
Isolation and Quantification of Murine Immune Cells
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The mice were sacrificed by cervical dislocation. Bone marrow, spleen and thymus were collected into tissue culture medium prepared as described previously [25 (link)]. Bone marrow cells were extracted by pressurized flow of buffer through dissected femurs and tibias. Single cell suspensions from spleen and thymus were prepared by passing the tissues through 70 μm nylon mesh filters (BD Biosciences). Red blood cells (RBC) in the spleen samples were removed by incubating splenocytes with RBC lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3). White blood cells were counted using the ViCELL cell counter (Beckham Coulter Inc.).
3
Isolation and Culture of Rabbit Adipose Tissue
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Adipose tissues were isolated from rabbits previously anesthetized with 40 mg/kg ketamine and 5 mg/kg xylazine intraperitoneal (IP). In each animal, a midline abdominal incision was made after which we harvested approximately 100 ml of adipose tissue from the perivesical region. The adipose tissue was minced in cold phosphate-buffered saline (PBS, pH=7.4), then digested with 0.1% collagenase type I (Sigma, Germany) for 90 minutes at 37˚C with modf erate shaking. The cell suspension was centrifuged at 1000 rpm for 4 minutes. We discarded the supernatant and suspended the pellet in 20 ml of the culture medium that consisted of 4.5 g/l glucose-Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Germany) supplemented with 15% regular fetal bovine serum (FBS, Gibco, Germany), and 1% penicillin-streptomycin (Gibco, Germany). The suspended cells were filtered through 75 μm nylon mesh filters (BD Biosciences, USA). Finally, 5 ml of the cell suspension was seeded into 60-mm culture dishes (Grainer, Germany). The cells were incubated at 37˚C in a humid environment with 5% CO2for 7 days. The medium was changed every day to remove any nonattached cells.
4
Multiparametric Flow Cytometry Analysis
5
Spleen Cell Suspension Preparation
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Spleen cell suspensions were prepared according to Salem et al. (2007) . Briefly, the spleen was homogenized by gently pressing the organ between the rough ends of two glass slides and filtered through nylon mesh filters (100 μm; BD Biosciences, CA, USA). The cells were suspended in PBS (Sigma Chemical Co., St. Louis, USA) and washed twice. Red blood cells were lysed with ammonium chloride_potassium buffer (ACK; Invitrogen, Carlsbad, CA, USA) and the remaining cells were again washed 3 times and counted. Viability was determined by trypan blue exclusion and consistently exceeded 90%.
6
Spleen Cell Isolation Protocol
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Single-cell suspensions were generated from spleen via gentle homogenization through nylon mesh filters (70 µM; Becton Dickinson, UK). Erythrocytes in spleens were lysed with ammonium chloride potassium (ACK) lysis buffer and the cell pellet was resuspended in DMEM medium supplemented with 10% v/v/FBS, 1% w/v/Penicillin/Streptomycin, 1 mM glutamine and 50 μM β-mercaptoethanol.
7
Isolation and Culture of Mouse Bone Marrow-Derived Macrophages
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Mouse bone marrow-derived macrophages cells (BMDMs) were isolated, as described previously (32 (link)). Briefly, bone marrow-cells were dissected from femurs and tibiae and plated into a complete media (RPMI 1640 medium (Sigma) supplemented with 10% v/v FBS (Life Technologies Ltd), 100 IU/mL penicillin 100 μg/mL streptomycin (Sigma), 50 ng/mL mouse colony-stimulating factor (MCSF) (Promega), and 50 μM β-mercaptoethanol) (Sigma) at 5 × 106 cells per 90 mm bacterial petri dish (Sterilin, UK) for 4 days. On day 4, 10 mL of complete media was added and incubated for 3 days. Adherent cells were then harvested with 5 mL of PBS supplemented with 5% v/v FBS and 2.5 mM EDTA. For splenocyte isolation, the spleen was homogenized and filtered through nylon mesh filters (70 μM; Becton Dickinson, UK) to generate a single-cell suspension. RBCs were lysed with ammonium chloride potassium (ACK) lysis buffer before the cell pellet was resuspended in DMEM medium supplemented with 10% v/v/FBS, 1% w/v/penicillin/streptomycin, 1 mM glutamine and 50 μM β-mercaptoethanol.
8
Isolation of Rat Cortical Synaptoneurosomes
9
Isolation of Rat Cortical Synaptoneurosomes
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Synaptoneurosomes were prepared from 3–4 month-old male Sprague-Dawley rats, as previously described37 (link), with minor modifications. Briefly, cortices were rapidly dissected and placed into chilled modified Krebs solution (mKrebs) containing (in mM): NaCl (118.5), KCl (4.7), KH2PO4 (1.18), NaHCO3 (24.9); MgSO4 (1.18), CaCl2 (2.5), D-glucose (10), HEPES (1), pH 7.4, and equilibrated with 95%O2/5%CO2. Cortices were homogenized in a 7-mL Kontes tissue Dounce homogenizer with ten strokes. Homogenized tissue was filtered through a 100-µm nylon mesh filter (BD Falcon) and the resulting suspension was filtered again through a 5-µm pore size Acrodisc syringe filter with a Supor membrane (Pall Life Sciences). The filtrate was then centrifuged at 1000 g for 15 min at 4 °C, washed once and centrifuged again. The pellet was resuspended in mKrebs and drugs were directly added to the pre-warmed (5 min, 37 °C) synaptoneurosome suspension for the indicated periods of time.
10
Isolation of Synaptoneurosomes from Rat Cortex
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Synaptoneurosomes were prepared from 2- to 3-month-old male Sprague-Dawley rats, as previously described (25 (link)), with minor modifications. Briefly, cortices were rapidly dissected and placed into chilled modified Krebs solution (mKrebs) containing (in mM): NaCl (118.5), KCl (4.7), KH2PO4 (1.18), NaHCO3 (24.9), MgSO4 (1.18), CaCl2 (2.5), d -glucose (10), HEPES (1), pH 7.4, and equilibrated with 95%O2/5%CO2. Cortices were homogenized in a 7-ml Kontes tissue Dounce homogenizer with 10 strokes. Homogenized tissue was filtered through a 100-μm nylon mesh filter (BD Falcon) and the resulting suspension was filtered again through a 5-μm pore size Acrodisc syringe filter with a Supor membrane (Pall Life Sciences). The filtrate was then centrifuged at 1000 × g for 15 min at 4°C, washed once, and centrifuged again. The pellet was resuspended in mKrebs and various drugs were directly added to the pre-warmed (5 min, 37°C) synaptoneurosome suspension for the indicated periods of time.
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