Organotypic brain slices were prepared using 6-8-day-old Wistar rats following the protocol originally described by Stoppini et al. [45 (link)]. Briefly, brains were extracted and swiftly placed in cold (4°C) dissection media consisting of Earle’s Balanced Salt Solution (Sigma-Aldrich) supplemented with D-glucose (6.1 g/L) and HEPES (6.6 g/L). The hemispheres were separated, and individual hippocampi were removed and immediately cut into 350 μm slices using a Mcllwain tissue chopper (Mickle). Cold dissection media was used to separate and rinse the slices before placing them onto Millicell-CM membranes (Sigma-Aldrich). Slices were maintained in culture medium consisting of 25% (vol/vol) Earle’s Balanced Salt Solution; 49% (vol/vol) minimum essential medium (Sigma-Aldrich); 25% (vol/vol) heat-inactivated horse serum (Sigma-Aldrich); 1% (vol/vol) B27 (Invitrogen, Life Technologies) and 6.2 g/l D-glucose (Sigma-Aldrich). Slices were incubated in a 5% carbon dioxide (CO2), humidified incubator at 37°C. Recordings were made after 6–14 days in culture.
Heat inactivated horse serum
Manufactured by Merck Group
Sourced in United States
Heat-inactivated horse serum is a laboratory reagent used to supplement cell culture media. It is produced by heating horse serum to inactivate any potential pathogens.
Automatically generated - may contain errors
Lab products found in correlation
Minimum essential medium (mem), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
D glucose, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mccoy s 5a medium, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Minimum essential medium (mem), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Earle s balanced salt solution, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Heat inactivated, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Cell Guidance Systems (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Nystatin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Alomone (1 mentions)
Recombinant human β ngf, by Alomone (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
27 protocols using heat inactivated horse serum
1
Organotypic Hippocampal Slice Culture
2
Differentiation of PC12 Cells with NGF
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PC12 cell line was obtained from Pasteur Institute of Iran (Tehran, Iran), and cultured on poly‐l‐ornithine (Sigma, USA) and laminin (Sigma)‐coated dishes in high‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% (v/v) heat‐inactivated horse serum (Sigma), 5% (v/v) heat‐inactivated fetal bovine serum (Gibco), and 100 U/ml penicillin–streptomycin (Gibco) at 37°C under a humidified atmosphere of 5% CO2. To induce differentiation, cells were treated for 7 days in medium containing 50 ng/ml of NGF‐β (Cell Guidance Systems, USA), 100 U/ml penicillin/streptomycin and 1% (v/v) horse serum. The half volume of differentiating medium was refreshed every 2 days.
3
Trichomonas vaginalis Infection of SiHa Cells
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T. vaginalis T106 was provided by Prof. J. K. Alderete (University of Texas, Health Science Center, TX, USA) and cultured in TYM medium consisting of 20% (w/v) trypticase peptone, 10% (w/v) yeast extract, 5% (w/v) Moltose monodydrate, 1% (w/v) L-cystein hydrochloride, 1% (w/v) ascorbic acid, 1% (w/v) K2HPO4, 1% (w/v) KH2PO4 (pH 6.2) with 10% heat-inactivated horse serum (Sigma-Aldrich, St. Louis, MO, USA) and penicillin-streptomycin (Gibco BRL) and incubated under 5% CO2 at 37 °C SiHa cells were infected with live T. vaginalis at multiplicities of infection (MOI) of 1 in the mixed-medium (DMEM without Antibiotic: TYM = 2:1) as previously described [25 (link)].
4
Establishing Multidrug-Resistant T-Cell Lymphoma Model
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L5178Y mouse T-cell lymphoma cells (PAR) (ECACC Cat. No. 87111908, obtained from FDA, Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as previously described by Cornwell et al. [59 (link)]. The ABCB1-expressing cell line L5178Y (MDR) was selected by culturing the infected cells with colchicine. The cells were cultured in McCoy’s 5A medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St Louis, MO, USA), 200 mM L glutamine (Sigma-Aldrich, St Louis, MO, USA), nystatin and a penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA) mixture in concentrations of 100 IU/L and 10 mg/L, respectively.
5
Cultivation of T. vaginalis Parasite
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The T016 strain of T. vaginalis was cultured in a glass, screw-capped tube containing Diamond's trypticase-yeast extract-maltose (TYM) medium (NAPCO, Winchester, VA, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich) in 5% CO2 at 37°C for 24 h [12 (link)]. Cultured parasites were monitored for motility and the viability of T. vaginalis was determined before each experiment using trypan blue staining (>99%).
6
Cultivation of Trichomonas vaginalis T016 Strain
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The T. vaginalis T016 strain was cultured according to previously reported methods [8 (link), 12 (link)]. Briefly, T. vaginalis T016 isolate was cultured in screw-capped glass tubes containing Diamond’s trypticase yeast-extract maltose (TYM) medium (NAPCO, Winchester, VA, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St Louis, MO, USA) in 5% CO2 at 37 ˚C for 24 h. The cultured parasites were monitored for motility, and their viability was determined before each experiment by staining with trypan blue (> 99%).
7
Establishing MDR1-expressing L5178Y Cell Line
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L5178 mouse T-cell lymphoma cells (PAR) (ECACC Cat. No. 87111908, obtained from FDA, Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as previously described by Cornwell et al. [47 (link)]. The ABCB1-expressing cell line L5178Y (MDR) was selected by culturing the infected cells with colchicine. The parental L5178 mouse T-cell lymphoma cells and the L5178Y human ABCB1-transfected subline were cultured in McCoy’s 5A medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St Louis, MO, USA), 200 mM L-glutamine (Sigma-Aldrich, St Louis, MO, USA), and a penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA) mixture in concentrations of 100 U/L and 10 mg/L, respectively. The cell lines were incubated at 37 °C, in a 5% CO2, 95% air atmosphere.
8
Culturing Trichomonas vaginalis Isolate T016
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The T. vaginalis T016 isolate was cultured in a glass, screw-capped tube containing Diamond's trypticase-yeast extract-maltose (TYM) medium (NAPCO, Winchester, Virginia, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St. Louis, Missouri, USA) in 5% CO2 at 37℃ for 24 hr. Cultured parasites were monitored for motility, and the viability of T. vaginalis was determined before each experiment using trypan blue staining (>99%).
9
Culturing and Treating Cell Lines for Parkinson's Research
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PC12 cells were cultured and treated with NGF as described previously.52 (link) For NGF treatment, the cells were cultured in RPMI 1640 medium (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 1% heat-inactivated horse serum (Sigma-Aldrich), penicillin/streptomycin, and 50 ng/ml recombinant human β-NGF (Alomone Labs, Jerusalem, Israel; or kindly provided by Genentech, South San Francisco, CA, USA) for 7–8 days, in a 7.5% CO2 atmosphere at 37 °C.
Neonatal rat superior cervical ganglion sympathetic neurons were cultured as previously described.19 (link), 53 (link)HEK293 cells were maintained in DMEM medium (Gibco Life Technologies) supplemented with 10% fetal calf serum (Gibco Life Technologies) and penicillin/streptomycin (Gibco Life Technologies) in a 5% CO2 atmosphere at 37 °C.
Human fibroblasts derived from six diagnosed AR-JP patients with PARK2 dysfunctional mutations and six control individuals were cultured in DMEM medium supplemented with 10% fetal calf serum and penicillin/streptomycin in a 5% CO2 atmosphere at 37 °C. These fibroblasts were obtained from the Neurology Service-Movement Disorders Biorepository (IDIBAPS, Hospital Clinic, Universitat de Barcelona, Barcelona, Catalonia, Spain). Fibroblasts were used between 5 and 6 passages in vitro.
Neonatal rat superior cervical ganglion sympathetic neurons were cultured as previously described.19 (link), 53 (link)HEK293 cells were maintained in DMEM medium (Gibco Life Technologies) supplemented with 10% fetal calf serum (Gibco Life Technologies) and penicillin/streptomycin (Gibco Life Technologies) in a 5% CO2 atmosphere at 37 °C.
Human fibroblasts derived from six diagnosed AR-JP patients with PARK2 dysfunctional mutations and six control individuals were cultured in DMEM medium supplemented with 10% fetal calf serum and penicillin/streptomycin in a 5% CO2 atmosphere at 37 °C. These fibroblasts were obtained from the Neurology Service-Movement Disorders Biorepository (IDIBAPS, Hospital Clinic, Universitat de Barcelona, Barcelona, Catalonia, Spain). Fibroblasts were used between 5 and 6 passages in vitro.
10
Mouse Sepsis Infection Model with Enterococci
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The mouse sepsis infection model was performed as described previously with some modifications [13 (link), 30 (link), 31 (link)]. Briefly, male BALB/c mice (weight range, 20–25 g; Harlan, Italy) were randomly separated into 4 groups of 6 and intraperitoneally injected 3 times with 200 µL of either normal rabbit serum, α-DHG-PpiC, or α-DHG-SagA 48 hours before, 24 hours before, and 4 hours after the bacterial challenge. Overnight cultures of E. faecalis type 2 and vancomycin-resistant E. faecium 11236/1 grown in brain heart infusion broth (Sigma-Aldrich) supplemented with 40% heat-inactivated horse serum (Sigma-Aldrich) were centrifuged, and the resulting pellets were resuspended in sterile PBS to achieve final concentrations of 109 bacteria/mL. Mice were first anaesthetized with 100 mg/g ketamine (Merial) and 12 mg/g xylazine (Bayer) via intraperitoneal injection. Aliquots of 100 µL from each strain suspension were injected intravenously into the corresponding group of mice. The animals were monitored twice per day before being euthanized by cervical dislocation 48 hours after the bacterial challenge. Kidneys and livers were aseptically removed, weighed, and homogenized in PBS for 120 seconds at high speed in a Stomacher (Pbi International). Serial dilutions were plated onto Enterococcus Selective Agar (Fluka Analytical) to determinate the number of colony-forming units.
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