Cells were collected from zebrafish scales and analyzed by flow cytometry as previously described (Kobayashi-Sun et al., 2020a (link)). Extracted scales were treated with Liberase TM (Roche) in PBS for 1 h at 37°C. The cells were then filtered through a 40 μm mesh and washed with 2% fetal bovine serum (FBS) in Hanks’ balanced salt solution (Wako) by centrifugation (300X g). Data were acquired on a FACS Aria III (BD Biosciences). Data analysis was performed using the Kaluza software (ver. 1.3, Beckman Coulter). The volume of fluid acquired per sec in FACS Area III was calculated by acquiring Accudrop beads (BD Bioscieces) whose concentrations were measured using a hemocytometer (Funakoshi). The absolute cell number was calculated based on the sample volume, acquisition events, acquisition times, and the percentage of each cell fraction.
Hank s balanced salt solution
Manufactured by Fujifilm
Sourced in Japan, United States
Hank's Balanced Salt Solution is a commonly used cell culture medium. It contains a balanced mixture of inorganic salts, including calcium, magnesium, potassium, and sodium, maintaining the osmotic balance and pH necessary for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Fujifilm (3 mentions)
Collagen 1, by Iwaki (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (1 mentions)
Liberase, by Roche (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Dmem high glucose, by Fujifilm (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Neonatal cardiomyocyte isolation kit, by Miltenyi Biotec (1 mentions)
Lew crlcrlj, by Charles River Laboratories (1 mentions)
Neonatal heart dissociation kit, by Miltenyi Biotec (1 mentions)
Deoxyribonuclease, by Nippon Gene (1 mentions)
Mgso4, by Kanto Chemical (1 mentions)
Cacl2, by Kanto Chemical (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
Polycarbonate membrane filter, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
20 protocols using hank s balanced salt solution
1
Zebrafish Scale Cell Isolation and Flow Cytometry
2
Primary Culture of Mouse Cerebellar Astrocytes
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Primary culture of mouse cerebellar astrocytes (25 (link), 26 (link)) was prepared using C57BL/6 pregnant mice (Japan SLC). Cerebella from postnatal day one of pups were dissected then digested in Hank’s balanced salt solution (Wako) containing 2.5% trypsin (Wako). Cerebellar tissue was incubated at 37°C for 30 min with continuous shaking. Mixed cerebellar tissue was centrifuge at 3500 rpm for 15 minutes at 4°C, the supernatant was discarded, and cells were resuspended in an astrocyte culture medium (high-glucose DMEM (Wako), 10% heat-inactivated FBS, and 1% penicillin/streptomycin). Cells were counted and 10–15 × 106 cells were plated on Collagen I coated 10–cm dishes (Iwaki, Japan), then incubated at 37°C in a CO2 incubator. The astrocyte culture medium was replaced with PBS on day 3 in vitro (DIV3). Oligodendrocyte precursor cells were removed by shaken the dishes for 2–5 minutes. Discard the supernatant then replaced with a fresh astrocyte culture medium. On DIV7, astrocytes were harvested and then plated on Collagen I coated 6 or 24 well dishes (Iwaki). Cells were then used for co-culture studies.
3
Isolation and Culture of Neonatal Rat Cardiomyocytes
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Hearts were removed from LEW/CrlCrlj (Charles River Laboratories Japan, Kanagawa, Japan) or LEW-Tg (Rosa-luc)11Jmsk (Jichi Medical University, Tochigi, Japan) neonatal rats aged 0–3 days old and washed three times with Hanks' Balanced Salt Solution (HBSS; Fujifilm Wako Pure Chemical, Tokyo, Japan) [[30] (link), [31] (link), [32] (link)]. The hearts were minced into pieces less than 2 mm in size, and 7–8 pieces of tissue were placed into a gentleMACS C tube and processed in a gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cardiomyocytes were collected using a Neonatal Heart Dissociation Kit (Milteny Biotec) and selected using a Neonatal Cardiomyocyte Isolation Kit (Milteny Biotec). The number of cardiomyocytes obtained was counted. The cells were seeded on culture dishes coated with fetal bovine serum (FBS; Thermo Fisher Scientific, Tokyo, Japan) and cultured under specific conditions for each experiment.
4
Primary Glial Cell Isolation Protocol
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Primary glial cells, mainly comprised of astrocytes and microglia, were obtained as previously described (36 ). Briefly, we isolated the cerebrum from postnatal day 2 mice of either sex in ice-cold Hanks’ balanced salt solution not containing Ca/Mg (FUJIFILM Wako Chemicals). The cerebrum was suspended in Hanks’ balanced salt solution containing 0.25% trypsin, 0.1 μl/ml deoxyribonuclease (Nippon Gene), 0.8 mM MgSO4 (Kanto Chemical Co), and 1.85 mM CaCl2 (Kanto Chemical Co) at 37 °C for 15 min. The obtained cell suspension was passed through a 100 μm cell strainer (Falcon) and centrifuged with a culture medium. The cell pellet was resuspended in the culture medium and seeded on cell culture plates. The culture medium was replaced at 4 days in vitro. The enrichment of glial cells was confirmed by immunocytochemistry using anti-glial fibrillary acidic protein and anti-Iba1 antibodies (Fig. S5 ).
5
Primary Culture of Mouse Astrocytes
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The animal experimentation protocol in the present study was approved by the Animal Care and Experimentation Committee, Gunma University (19-024, 17 December 2018), and all efforts were made to minimize animal suffering and the number of animals used.
A primary culture of mouse cerebral cortex astrocytes was prepared as previously described (29 (link), 30 (link)) with slight modifications. A pregnant C57BL/6 strain mice were purchased from Japan SLC (Hamamatsu, Japan). Briefly, postnatal day 1 mouse cerebral cortices were dissected and digested with 2.5% trypsin (Wako, Japan) in Hank’s balanced salt solution (Wako) for 30 min with continued shaking at 37°C. Cells were resuspended in an astrocyte culture medium (high-glucose DMEM, 10% heat-inactivated FBS, and 1% penicillin/streptomycin), and 10–15 million cells were plated on 10-cm dishes coated with Collagen I (Iwaki, Japan). Cells were incubated at 37°C in a CO2 incubator. On day 3 in vitro (DIV3), astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako), and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.
A primary culture of mouse cerebral cortex astrocytes was prepared as previously described (29 (link), 30 (link)) with slight modifications. A pregnant C57BL/6 strain mice were purchased from Japan SLC (Hamamatsu, Japan). Briefly, postnatal day 1 mouse cerebral cortices were dissected and digested with 2.5% trypsin (Wako, Japan) in Hank’s balanced salt solution (Wako) for 30 min with continued shaking at 37°C. Cells were resuspended in an astrocyte culture medium (high-glucose DMEM, 10% heat-inactivated FBS, and 1% penicillin/streptomycin), and 10–15 million cells were plated on 10-cm dishes coated with Collagen I (Iwaki, Japan). Cells were incubated at 37°C in a CO2 incubator. On day 3 in vitro (DIV3), astrocyte culture medium was replaced with phosphate-buffered saline (PBS). Dishes were then shaken by hand for 30–60 s until only the adherent monolayer of astrocytes was left. The PBS was then replaced with a fresh astrocyte culture medium. Astrocytes were harvested on DIV7 using 0.25% trypsin 1 mM disodium EDTA (Wako), and then plated on 12 or 24 well dishes. Cells were used for cell invasion assay or F-actin staining.
6
Isolation and Differentiation of Peripheral Blood Monocytes
7
Cytotoxicity of Magnetite Nanoparticles
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CT26 cells in 100 μL of RPMI medium were seeded at 3 × 103 cells/well in 96‐well plates and incubated at 37°C for 24 h. After preculture, 10 μL of magnetite dispersion was added to each well to obtain final concentrations of 10, 100, and 1000 μg/mL, and incubated for 24 h. Thereafter, the cells were washed twice with 100 μL of Hanks' Balanced Salt Solution (Fujifilm Wako Pure Chemical), and 100 μL of RPMI1640 medium was added. Next, 10 μL of Cell Counting Kit 8 (Dojindo) was added and incubated for 2 h. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader (iMark Microplate Absorbance Reader; Bio‐Rad Laboratories). The absorbance of cells cultured without MNPs was used as a control. The absorbance of RPMI1640 medium without cells was used as a blank.
8
Host Cell Invasion by Parasites
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Parasites were collected by scraping the cell monolayer and released from host cells by passage through a 21-gauge needle. Extracellular parasites were filtered onto a polycarbonate membrane filter (3.0-μm pore size; Millipore, Bedford, MA, USA) and washed with Hanks' balanced salt solution (Wako) containing 0.1 mM EGTA and 10 mM HEPES. HFFs were cultivated on a cover slip (Matsunami Glass, Osaka, Japan) in 12-well plates for 48 h before the experiment. Parasites were pretreated with 1 μM cytochalasin D (Sigma-Aldrich) at room temperature for 10 min. 1.0×108 parasites were inoculated on host cells and cultured at 37°C for 60 min in the presence of 1 μM cytochalasin D. After washing with 1 mM D-PBS, the culture was fixed with 4% paraformaldehyde at 4°C for 20 min.
9
Cell Sorting by p75NTR and Cell Cycle
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Cultured cells were washed once with phosphate-buffered saline (PBS), then dissociated from culture plates by using 0.25% trypsin EDTA (1X) and phenol red (Life Technologies, Carlsbad, CA, USA) and were centrifuged at 300 × g for 10 min. Single cells were resuspended in PBS containing 2% FCS and allophycocyanin (APC)-conjugated human CD271 (LNGFR) antibody (miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or a compared isotype control were incubated in the dark at 4°C for 30 min. After washing twice with PBS containing 2% FCS, the cells were resuspended in hank's balanced salt solution (Wako Pure Chemical Industries), were treated with Vybrant® DyeCycle™ Violet stain (DCV; Invitrogen/molecular Probes, Eugene, OR, USA) and were mixed well. Next, the cells were incubated at 37°C for 30 min, protected from light. Cell samples by using a flow cytometer (BD FACSAria™ II; BD Biosciences, San Jose, CA, USA) were sorted into the following four fractions: i) p75NTR-positive cells in the G0-G1 phase (p75NTR-positive/G0-1); ii) p75NTR-positive cells in the S-G2-M phase (p75NTR-positive/S-G2-M); iii) p75NTR-negative cells in the G0-G1 phase (p75NTR-negative/G0-1); iv) p75NTR-negative cells in the S-G2-M phase (p75NTR-negative/S-G2-M). Each population was evaluated as follows.
10
Inhibitor Compounds for Cell Death Pathways
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Lapatinib, FTY720, hydroxychloroquine (HCQ), U18666A and gefitinib were purchased from Cayman Chemical Company and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM to obtain stock solutions. Abemaciclib and z-VAD-FMK, pan-caspase inhibitors, were purchased from AdooQ BioScience. E64d and pepstatin A, inhibitors of lysosomal proteases, were purchased from Peptide Institute, Inc. Necrostatin-1, a specific inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), was purchased from Enzo Life Sciences, Inc., and N-acetyl-L-cysteine, staurosporine, bafilomycin A1 and Hanks' balanced salt solution (HBSS) were obtained from FUJIFILM Wako Pure Chemical Corporation. Hoechst 33342 was obtained from Nacalai Tesque, Inc., and DAPI was purchased from Sigma-Aldrich (Merck KGaA).
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