Oregon GreenTM 488–conjugated gelatin-coated (G13186, Thermo Fisher Scientific) coverslips were prepared as previously described81 . In short, coverslips (12 mm diameter, No. 1 thickness; 631-0666, VWR International) were precleaned in 4% nitric acid for 30 min. After washing, the coverslips were coated with 50 µg ml−1 poly-L-lysine (P7890, Merck/Sigma) for 30 min, washed in PBS, and fixed with cold 0.5% glutaraldehyde (G5882, Merck/Sigma) in PBS for 15 min on ice. Subsequently, the coverslips were washed in PBS and coated for 10 min with preheated (44 °C) 10 mg ml−1 unlabelled or Oregon GreenTM 488–conjugated gelatin/2% sucrose in PBS. After coating, the coverslips were washed with PBS and incubated in 5 mg ml−1 sodium borohydride (452882, Merck/Sigma) for 15 min. The coverslips were then washed with PBS, sterilized with 70% ethanol, and equilibrated in serum-containing medium for 1 h at 37 °C before addition of cells.
P7890
Manufactured by Merck Group
Sourced in United States
The P7890 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating samples at speeds up to 15,000 rpm and can accommodate a variety of rotor sizes. The centrifuge features a digital display for time and speed control, as well as safety mechanisms to ensure proper operation.
Automatically generated - may contain errors
Lab products found in correlation
G5882, by Merck Group (1 mentions)
Mpeg sva 20k, by Nanocs (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
F6886, by Merck Group (1 mentions)
S2532, by Merck Group (1 mentions)
M7898, by Merck Group (1 mentions)
C6645, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Tyrisin, by Merck Group (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Sodium carbonate, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate isomer 1, by Merck Group (1 mentions)
Calcium chloride dihydrate, by Thermo Fisher Scientific (1 mentions)
Milli q purification system, by Merck Group (1 mentions)
H5515, by Merck Group (1 mentions)
P4005, by Merck Group (1 mentions)
6 protocols using p7890
1
Gelatin-coated Coverslips for Cell Culture
2
Sterically Stabilized Microspheres for Chlamydomonas Experiments
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CR cells are synchronously grown in 12:12 hr light:dark cycle in Tris-Acetate-Phosphate (TAP + P) medium. This culture medium contains divalent ions such as Ca2+, Mg2+, SO42- which decrease the screening length of the 200 nm negatively charged microspheres, thereby promoting inter-particle aggregation and sticking to glass surfaces and CR’s flagella. Therefore, the sulfate latex microspheres (S37491, Thermo Scientific) are sterically stabilized by grafting long polymer chains of polyethylene glycol (mPEG-SVA-20k, NANOCS, USA) with the help of a positively charged poly-l-lysine backbone (P7890, 15–30 kDa, Sigma) (Mondal et al., 2020 (link)). In addition, the coverslip and slide surfaces are also cleaned and coated with polyacrylamide brush to prevent non-specific adhesion of microspheres and flagella to the glass surfaces, prior to sample injection (Mondal et al., 2020 (link)).
3
Purification and Culture of Rat Sciatic Nerve Schwann Cells
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SCs were cultured and purified as previously described (Gu et al. 2014 (link)). Sciatic nerves were harvested from neonatal rats (1–2 days), cut into small pieces and enzymatically treated with 1% collagenase (17018-029, Gibco, Carlsbad, CA, USA) and 0.125% trypsin (25200-056, Gibco). The cell mixture was then resuspended in DMEM (11965-092, Gibco), 10% FBS (12483-020, Gibco), and penicillin–streptomycin (PS; 15140122, Thermo Fisher Scientific, Cleveland, OH, USA) using a 400-mesh cell screen to make a single-cell suspension, seeded in 50 μg/ml poly-d -lysine (PDL; P-7890, Sigma, St Louis, MO, USA) coated dishes with 10 mM cytosine arabinoside (C6645, Sigma) and 10% FBS in DMEM for 1 day to remove fibroblasts. Afterwards, the medium was changed to supplemented with 5 μM forskolin (F6886, Sigma), 2 ng/ml Neuregulin 1 (Nrg1; 396-HB-050, R&D, MN, USA) and 10% FBS in DMEM. After 48 h, cells were incubated with anti-Thy1 antibody (1:1000, M7898, Sigma) to remove remaining contaminating fibroblasts. SC purity was assessed by S100β (1:500, S2532, Sigma) immunostaining. Passage 2 SCs with > 95% purity were used for all cell experiments.
4
Dissociation of Rat DRG Neurons
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DRG neurons from SD rats (60–80 g) were dissociated as previously described [24 (link)]. Briefly, L4–L6 DRGs were excised in ice-cold DMEM/F12 medium (10565018, GIBCO, USA) and mechanically dissociated. After digested with tyrisin (T9201, Sigma, USA) and collagenase (C9891, Sigma, USA) for 30 min in 37 °C, DRG neurons were seeded onto cover slips coated with poly-l -lysine (P7890, Sigma, USA) in a humidified atmosphere (5% CO2, 37 °C) for up to 4 h and then were used for patch-clamp recording.
5
Biopolymer Characterization and Functionalization
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Calcium chloride dihydrate
(Acros Organics, 10158280), sodium carbonate (Acros Organics, 10577182),
sodium chloride (Fisher BioReagents, 10316943), EDTA [ethylenediaminetetraacetic
acid (Fischer Scientific, 10335460)], chondroitin sulfate A 50 kDa
(Creative PEGWorks, CS-114), fluorescein isothiocyanate–dextran
sulfate sodium salt 40 kDa (Sigma, 51923), hyaluronic acid 50 kDa
(Creative PEGWorks, HA-102), heparin sodium salt from porcine intestinal
mucosa 10–12 kDa (Sigma, H5515), collagen type1 from rat tail
115–130 and 215–235 kDa (Sigma, 08–115), dextran
amine 70 kDa (Creative PEGWorks DE-664), poly-l -lysine hydrobromide
15–30 kDa and 150–300 kDa (Sigma-Aldrich, P7890 and
P1399), protamine from salmon (Grade IV) 5–10 kDa (Sigma, P4005),
fluorescein isothiocyanate-hyaluronic acid 50 kDa (Creative PEGWorks),
fluorescein isothiocyanate-chondroitin sulfate A 50 kDa (Creative
PEGWorks), fluorescein isothiocyanate isomer I was also from Sigma,
and ethanol (absolute, >99%) was purchased from Fisher. Tris-buffered
saline (10× Tris) pH 7.4 (Alfa Aesar, J60764) containing 250
mM Tris, 1.37 M sodium chloride, and 27 mM potassium chloride has
been used. The water used in the experiments was prepared using a
Millipore Milli-Q purification system and had a resistivity higher
than 18.2 M Ω cm.
(Acros Organics, 10158280), sodium carbonate (Acros Organics, 10577182),
sodium chloride (Fisher BioReagents, 10316943), EDTA [ethylenediaminetetraacetic
acid (Fischer Scientific, 10335460)], chondroitin sulfate A 50 kDa
(Creative PEGWorks, CS-114), fluorescein isothiocyanate–dextran
sulfate sodium salt 40 kDa (Sigma, 51923), hyaluronic acid 50 kDa
(Creative PEGWorks, HA-102), heparin sodium salt from porcine intestinal
mucosa 10–12 kDa (Sigma, H5515), collagen type1 from rat tail
115–130 and 215–235 kDa (Sigma, 08–115), dextran
amine 70 kDa (Creative PEGWorks DE-664), poly-
15–30 kDa and 150–300 kDa (Sigma-Aldrich, P7890 and
P1399), protamine from salmon (Grade IV) 5–10 kDa (Sigma, P4005),
fluorescein isothiocyanate-hyaluronic acid 50 kDa (Creative PEGWorks),
fluorescein isothiocyanate-chondroitin sulfate A 50 kDa (Creative
PEGWorks), fluorescein isothiocyanate isomer I was also from Sigma,
and ethanol (absolute, >99%) was purchased from Fisher. Tris-buffered
saline (10× Tris) pH 7.4 (Alfa Aesar, J60764) containing 250
mM Tris, 1.37 M sodium chloride, and 27 mM potassium chloride has
been used. The water used in the experiments was prepared using a
Millipore Milli-Q purification system and had a resistivity higher
than 18.2 M Ω cm.
6
Immobilization of E. coli DH10B
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The immobilization of the E. coli (DH10B) was performed in following steps. First, the glass-bottom Petri dish was coated with 1 µg/ml PLL (P7890; Sigma-Aldrich) in milli-Q water for 30 min and washed thrice with milli-Q water. E. coli was harvested by three cycles of centrifuging (10,000 relative centrifugal force), discarding supernatant, and resuspending in milli-Q water. Bacteria solution after density adjustment was transferred onto the PLL-coated Petri dish and incubated for 30 min at room temperature. The E. coli–immobilized PLL surface was washed thrice with milli-Q water, and we continued to perform SNS coating according to the above-described method.
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