Primary neonatal rat astrocyte conditioned media was prepared as previously described [6 (link), 7 (link)]. For microglia conditioned media, primary neonatal rat glial cells were isolated and cultured as previously described [18 (link)]. Upon culture confluence, cells were dissociated using 0.25% Trypsin EDTA (Thermo Fisher Scientific) and microglia were isolated by fluorescence activated cell sorting (FACS) for CD11b/c positive cells. The sorted cells were cultured on poly-d-lysine coated plates (Sigma-Aldrich, 10 μg/mL) in DMEM with 10% FBS, and purity was confirmed by immunofluorescence and the absence of staining for astrocytes with a GFAP antibody. Conditioned media was collected after 48 h of incubation with microglia cultures of 70% or greater confluence. Mouse embryonic fibroblasts (MEFs) were purchased from ATCC and cultured in DMEM with 10% FBS. Conditioned media was collected after 48 h of incubation with the MEF cultures of 70% or greater confluence. All conditioned media were filtered through 0.4 μM filters prior to use to remove any dead cells or cellular debris.
Poly d lysine coated plates
Manufactured by Merck Group
Sourced in Germany
Poly-d-lysine-coated plates are a type of laboratory equipment used for cell culture applications. The plates have a surface that is coated with the synthetic amino acid polymer poly-d-lysine, which enhances cell attachment and growth. These plates are commonly used in various cell-based experiments and assays.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Proliferation supplement, by STEMCELL (1 mentions)
Shh c24ii, by R&D Systems (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Hemoglobin, by Merck Group (1 mentions)
M2 flag conjugated magnetic beads, by Merck Group (1 mentions)
Actn05, by Abcam (1 mentions)
Anti mouse nxa931, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
5 protocols using poly d lysine coated plates
1
Isolation and Conditioning of Neonatal Rat Astrocytes and Microglia
2
Isolation and Polarization of Rat Microglia
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Primary microglia were isolated from adult male Sprague-Dawley rat brains, as described previously [25 (link)]. The cells were cultured (1.2 x 105 cells per well in 2 mL of medium) in six-well poly-d -lysine-coated plates (Sigma-Aldrich, P0296) and grown in microglia culture medium (Dulbecco's modified Eagle's medium/F-12 Glutamax; Gibco, 10565042) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin and 10 ng/mL rat recombinant carrier-free macrophage colony stimulating factor (M-CSF; Peprotech, 400-28) at 37°C with 5% CO2. Half of the medium was changed every three days. M0, M1 and M2 microglia were polarized as described previously [26 (link)].
3
Neurosphere Culture and In Vitro Treatments
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For neurosphere cultures, sorted cells were plated on poly-D-lysine-coated plates (Sigma) at a density of 1–3 × 103 cells/well in 96-well tissue culture plates (TRP). The culture medium was composed of NeuroCult medium complemented with the proliferation supplement (STEMCELL Technologies), 2 μg/mL heparin, 20 ng/mL EGF, and 10 ng/mL fibroblast growth factor 2. Seven days after plating, the neurospheres were counted and their area measured with ImageJ software. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. In vitro treatments with SHH (SHH C24II; R&D 1845-Sh) were done at 10 nM and with MRT-83 at 1 μM.
4
Primary Neuronal Cell Cultures for SAH Research
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Primary neuronal cell cultures were prepared from the cortex of fetal C57BL/6 J mice at 13 days of gestation as a modification of a previously described method18 (link). Cells were seeded in poly-d -lysine-coated plates (Sigma, Darmstadt, Germany) with neurobasal medium containing 0.5 mM GlutaMax-I (Gibco Company, USA) and 2% B27 supplement (Gibco Company, USA). The medium was replaced 4 h after seeding. Subsequently, half of the medium was replaced every 3 days following seeding. For the in vitro SAH model, cells were incubated with hemoglobin (Millipore Sigma, Darmstadt, Germany) for 24 h at a concentration of 25 μM. Primary neuronal cell cultures were randomly assigned to different groups.
5
PTRN-1 Protein Detection and Co-Immunoprecipitation
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Worm protein samples were prepared by boiling 10 μl pellets of mixed-stage worms in SDS-mercaptoethanol solution. We detected PTRN-1 using antibody OD208A (Wang et al., 2015 (link)); we used the anti-actin antibody ACTN05 (Abcam, Cambridge, MA, C4) as a loading control. We used HRP-linked anti-rabbit IgG NA934V and anti-mouse NXA931 as secondary antibodies (GE Healthcare Lifesciences) at 1:1000 dilution in TBS, and added SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA). The blot result was replicated once.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl2) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.
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