The SGCs of DRG (C1-T2) from neonatal Sprague-Dawley rats were prepared as follows. Briefly, 1- to 3-day-old neonatal rats were anesthetized, and DRGs were harvested in ice-cold Hank’s balanced salt solution (HBSS). DRGs were removed with fine dissecting forceps from the inner side of each half of the dissected vertebrae together with the dorsal and ventral roots and attached spinal nerves. After removing the attached nerves and the surrounding connective tissue, DRGs were incubated with trypsin (2.5 mg/mL; type III, Sigma), collagenase (1.0 mg/mL; type IA, Sigma), and DNase (0.1 mg/mL; type IV, sigma) at 37°C in a shaking bath for 15 min. Subsequently, 10% fetal bovine serum (FBS) was added to stop enzymatic digestion. After centrifuging (5 min, 1000 rpm), the remaining ganglia were dissociated into single cells by trituration with heat-polished Pasteur pipettes and passed through nylon mesh with a pore diameter size of 100 μm. Isolated cells were suspended in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco by Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin. These cells were seeded on uncoated 35 mm dishes at 37°C with 5% CO2 for up to 3 days. The media were completely changed every day. Before calcium fluorescence imaging, DRG SGCs were cultured with or without baicalin (10 μM) for 24 h. The experiments were performed at room temperature (20–30°C).
Type 4
Manufactured by Merck Group
Sourced in United States
Type IV is a laboratory equipment designed for general use in scientific research and analysis. It is a multi-purpose device that can perform various functions to support laboratory workflows. The core function of Type IV is to provide a controlled environment for conducting experiments and tests. Its specifications and capabilities are tailored to meet the needs of a wide range of scientific disciplines.
Automatically generated - may contain errors
Lab products found in correlation
Dnase, by Merck Group (3 mentions)
Type ia, by Merck Group (2 mentions)
Type 3, by Merck Group (2 mentions)
Collagenase, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fluorescence activated cell sorting (facs), by Beckman Coulter (1 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
H 2kd, by BD (1 mentions)
H2 kb, by BD (1 mentions)
Faser kit, by Miltenyi Biotec (1 mentions)
Rbc lysis buffer, by Merck Group (1 mentions)
Pervanadate, by Merck Group (1 mentions)
Rat tail collagen type 1, by Merck Group (1 mentions)
Type 2, by Merck Group (1 mentions)
Type 2 s, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
70 μm strainer, by BD (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Ov110, by Olympus (1 mentions)
Attune cytometer, by Thermo Fisher Scientific (1 mentions)
10 protocols using type 4
1
Isolation and Culture of Rat DRG SGCs
2
Isolation of Female Germline Stem Cells
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Ovaries from a total of 500 female mice (Ddx4-Cre; mT/mG mice, aged 3–5 days) were collected, washed with ice-cold phosphate-buffered saline (PBS), and cut into small pieces. Two-step enzymatic isolation of FGSCs was performed, as described previously [12 (link)]. Briefly, the mouse ovarian tissue was treated with 1 mg/ml collagenase (Type IV; Sigma), followed by 0.05% trypsin and 1 mM EDTA digestion at 37°C to dissociate cells. After passing through a 13-μm nylon cell filter, the cells were suspended in PBS and subjected to fluorescence-activated cell sorting (FACS), according to the manufacturer's instructions (Beckman Coulter), to sort GFP-positive cells. Then, GFP-positive cells were suspended in PBS and plated in 35 mm cell culture plates precoated with mouse laminin (4. 4 μg/cm2). After incubated for 45min at 37°C, unbound cells were removed from bound cells by pipetting.
3
Murine Alloantibody Detection and K^d Expression
4
Collagen and Pervanadate Stimulation
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Rat tail collagen type I (Sigma-Aldrich, Saint Louis, MO, USA) and type IV (Sigma-Aldrich, Saint Louis, MO, USA) were used to stimulate cells at a final concentration of 20 µg/mL. Pervanadate (Sigma-Aldrich, Saint Louis, MO, USA) was prepared freshly, as previously described [24 (link)].
5
Cardiac Myocyte Isolation and Culture
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CBs were incubated (12 min; 37 °C) in nominally Ca2+- and Mg2+-free Tyrode’s solution (pH = 7.2) containing collagenase (2.5 mg/mL, type IV, Sigma) and bovine serum albumin (BSA; 6 mg/mL, Fraction V, Sigma). After removing the solution, the CBs were incubated for 17 min period in a new Tyrode solution containing trypsin (1 mg/mL, type II, Sigma) and BSA (6 mg/mL). The trypsin-containing solution was removed, and the tissues were mechanically disrupted by aspiration through a P1000 pipette in 2 mL of culture medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin/streptomycin/fungizone. After centrifugation (2000 rpm, 7 min), the supernatant was discarded, and the cell pellet was resuspended in 100 µL of fresh culture medium. Dispersed cells were plated as 10–20 µL drops on small poly-L-lysine-coated coverslips kept in 12 well plates and maintained in a humidified incubator (37 °C; 5% CO2 in air). Once the cells attached (45 min after plating), 1.5 mL of DMEM was added into each well to maintain the cells until use (16–24 h later).
6
Isolation of Satellite Glial Cells from Rat DRG
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The rats were anesthetized with urethane (1.2 g/kg, intraperitoneally, i.p.). The DRG were isolated from the rats and transferred immediately into Dulbecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4 and 340 mosmol/kg. After removal of the attached nerves and surrounding connective tissues, the DRGs were minced with dissecting spring scissors and incubated with trypsin (0.5 mg/ml; type III, Sigma), collagenase (1.0 mg/ml; type IA, sigma) and DNase (0.1 mg/ml; type IV, Sigma) in 5 ml of DMEM at 35 °C in a shaking bath for 35–40 min. Then, soybean trypsin inhibitor (1.25 mg/ml; type II-S, Sigma) was added to stop the enzymatic digestion. The isolated non-neuronal cells (satellite glial cells) were transferred into a 35-mm culture dish and kept stationary for 30 min [39 (link)]. The experiments were performed at room temperature (20–30 °C).
7
Multimodal Imaging and Flow Cytometry for Tumor Analysis
8
Isolation of Porcine Fragilis+ Fetal Germ Stem Cells
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Porcine FGSCs (pFGSCs) were isolated using a two-step enzymatic digestion method, as described previously. Briefly, ovaries collected from 8- and 32-week-old Banna mini-pigs were enzymatically dissociated in a buffer containing collagenase (1 mg/ml, Type IV; Sigma-Aldrich, MO, USA) for 30 min with gentle agitation in a 37°C water bath, centrifuged and then washed once with phosphate-buffered saline (PBS). The ovarian tissues were further digested in 1 mM EDTA and 0.01% trypsin at 37°C for 15–20 min. The dissociated ovarian cell suspensions were filtered through a sterile cell strainer (70 μm nylon mesh). pFGSCs were enriched by immunomagnetic isolation of Fraglis+ cells. Goat anti-rabbit IgG microbeads (MiltenyiBiotec, Germany) were precoated with an anti-Fragilis primary antibody (Abcam, ab15592, UK). Fraglis+ cells were isolated by magnetic separation, according to the manufacturer’s instructions.
9
Confocal Imaging of Cell Cultures
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10
Isolation of Metastatic Tumor Cells
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Fresh metastatic tumor tissue recovered from the groin was minced with a scalpel. The tissue fragments were treated with collagenase (2 mg/mL, Type IV, Sigma) and DNA:se (100 mg/mL, Sigma) in serum-free culture medium for 4 h at 37°C under agitation. The cell suspension was pelleted by centrifugation, washed once
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