This protocol results in sample amounts suitable for several analyses. From 2 ml of culture of OD578 ≈ 10 about 300 µl purified PGN are gained. Spin down 2 ml of an overnight culture in a 2 ml microcentrifuge tube (Eppendorf) for 5 min at 10,000 rpm. Alternatively: Spin down 2 × 2 ml of a culture with a lower OD. Resuspend the pellet in 1 ml solution A (1 M sodium chloride) and boil the suspension for 20 minutes at 100°C in a heating block. [Δ CRITICAL STEP 1: Sometimes, NaCl treatment is not sufficient for peptidoglycan isolation from the cells. Use 0.25% SDS solution in 0.1 M Tris/HCl (pH 6.8) instead. SDS has to be washed out thoroughly after boiling. Make sure the samples are boiling at 100°C. Bad isolation results are mostly caused by too low heat.] Spin down the suspension (5 min at 10,000 rpm), wash it at least twice with 1.5 ml ddH2O and resuspend the pellet in 1 ml ddH2O. Put the sample to a sonifier waterbath for 30 minutes. Add 500 µl of solution B (15 μg/ml DNase and 60 μg/ml RNase in 0.1 M TRIS/HCl, pH 6.8) and incubate for 60 minutes at 37°C in a shaker. Add 500 µl of solution C (50 μg/ml trypsin in ddH2O) and incubate for additional 60 minutes at the same conditions. To inactivate the enzymes boil the suspension for 3 minutes at 100°C in a heating block, then spin the sample down (5 min at 10,000 rpm) and wash it once with 1 ml ddH2O. To release WTA resuspend the pellet in 500 µl of 1 M HCl (ready-to-use solution from Applichem) and incubate for 4 h at 37°C in a shaker. Spin down the suspension (5 min at 10,000 rpm) and wash with ddH2O until the pH is 5–6. Afterwards, resuspend the pellet in 100–250 µl digestion buffer (12.5 mM sodium dihydrogen-phosphate, pH 5.5) to an OD578 of 3.0 and add 1/10 volume of mutanolysin solution (5.000 U/ml of mutanolysin in ddH2O). [Δ CRITICAL STEP 2: If OD578 is too high the sample is too concentrated. Therefore, the digestion with mutanolysin might be disturbed. Measurement of OD is tricky, because peptidoglycan sinks to the bottom. Mix the suspension carefully with a pipette and measure OD rapidly.] Then incubate the sample for 16 h at 37°C (150 rpm shaking). Inactivate mutanolysin by boiling (100°C) for 3 min. Spin the sample down (5 min at 10,000 rpm) and use the supernatant. Before applying the sample to the UPLC system, MurNAc has to be reduced to NAc-muraminitol. Therefore, add 50 µl of reduction solution (10 mg/ml sodium borohydrate in 0.5 M borax in ddH2O at pH 9.0; both reagents were purchased from Merck) and incubate the sample for 20 minutes at room temperature. Stop the reaction with 10 µl phosphoric acid (98%). The resulting pH must be between 2 and 3. Then analyze the sample by UPLC/MS or HPLC/MS.
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Borax
Borax
Borax, a naturally occurring mineral compound, is a versatile chemical with a wide range of applications in industry and research.
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Most cited protocols related to «Borax»
borax
Buffers
Cells
deoxyribonuclease B
Digestion
Enzymes
High-Performance Liquid Chromatographies
isolation
mutanolysin
Peptidoglycan
Phosphoric Acids
ribonuclease M
Saline Solution
sodium borohydride
Sodium Chloride
sodium phosphate, monobasic
Tromethamine
Trypsin
The four cpDNA isolation methods used in our study were described as below:
A. Modified high salt method (Figure 5 )
Reagents
Buffer A (PH 3.8)
1.25 M NaCl, 0.25 M ascorbic acid, 10 mM sodium metabisulfite, 0.0125 M Borax, 50 mM Tris-HCl (pH 8.0), 7 mM EDTA, 1% PVP-40 (w/v), 0.1% BSA (w/v), 1 mM DTT;
Buffer B (PH 8.0)
1.25 M NaCl, 0.0125 M Borax, 1% PVP-40 (w/v), 50 mM Tris-HCl (PH 8.0), 25 mM EDTA, 0.1% BSA (w/v), 1 mM DTT;
Buffer C
100 mM NaCl, 100 mM Tris-HCl (PH 8.0), 50 mM EDTA, 1 mM DTT;
Both BSA and DTT were added just before the start of the experiment.
Chloroplast isolation
All the following steps were carried out at 0°C if not otherwise stated.
1. Prior to extraction, about 20 g (fresh weight) leaves were collected and kept in dark for 48 to 72 hours at 4°C to decrease starch level stored in the leaves.
2. The leaves were nervure-removed, cut into pieces (∼1 cm) and homogenized in 400 ml ice-cold buffer A for 30 seconds. Filter the homogenate into centrifuge bottles using two layers of Miracloth (Merck) by softly squeezing the cloth.
3. Centrifuged the homogenate (200 g, 20 min). The nucleus pellet and cell-wall debris were discarded.
4. Repeated the centrifugation once again. The supernatant included chloroplasts suspended in it.
5. Centrifuged the supernatant at a higher centrifugal force of 3500 g for 20 min, the resulting pellet were chloroplast pellet with some contamination of nuclear DNAs.
6. Added 250 ml Buffer B to the pellet and suspend it gently using a paintbrush to wash the nuclear DNAs attaching to the chloroplast cytomembrane. Then centrifuge with 3500 g for 20 min and discard the supernatant.
7. Re-suspended the pellet with 250 ml Buffer B again and centrifuged (3750 g for 20 min) to gain the purified chloroplasts.
Chloroplast DNA isolation
8. Added 8 ml Buffer C, 1.5 ml 20% SDS, 20 µl β-Me, 30 µl Proteinase K (10 mg/ml) to the purified chloroplast pellet and incubate at 55°C for at least 4 hours or overnight. The chloroplasts would be fully lysed.
9. Put the centrifuge bottles on ice for 5 minutes, add 1.5 ml 5 M KAc (PH 5.2) and continue to freeze for 30 minutes. Then 10000 g centrifuge 15 min, discarding the pellet.
10. Extracted the supernatant with an equal volume of saturated phenol and chloroform∶isoamyl-alcohol (24∶1) in the centrifugation of 10000 g 20 min for twice.
11. Added an equal volume of isopropyl alcohol (about 10 ml) to the upper clear aqueous phase. Then put the centrifuge bottles in the −20°C for 1 hour or overnight.
12. Centrifuged the aqueous phase at 10000 g for 20 min. The cpDNA pellet was washed repeatedly with ethanol (70%, 96%), air-dried, and re-dissolved in 50 µl TE buffer.
13. Treated the cpDNA sample with 2 µl RNAse and visualize the DNA band on a 0.8% agarose gel.
B. high salt method [13] (link)
Reagents
Cold isolation buffer: 1.25 M NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.1% BSA (w/v), 0.1% β-mercaptoethanol (v/v).
Chloroplast isolation
All the following steps were carried out at 0°C if not otherwise stated.
1. Prior to extraction, about 20 g (fresh weight) leaves were collected and kept in dark for 48 to 72 hours at 4°C in order to decrease starch level stored in the leaves.
2. The leaves were cut into pieces (∼1 cm) and homogenized in 100 ml ice-cold Cold isolation buffer for 30 seconds. Filter the homogenate into centrifuge bottles using two layers of Miracloth (Merck) with softly squeezing the cloth.
3. Centrifuged the homogenate (3000 g, 10 min).
4. Resuspended the chloroplast pellet in 30 ml cold isolation buffer, and repellet the chloroplasts (3000 g, 10 min).
5. Resuspended the final chloroplast pellet in 10 ml cold isolation buffer.
Chloroplast DNA isolation
6. Added 1/10 volume of 10% CTAB to lyse the chloroplasts. Incubate at 55°C for 1 to 2 hours.
7. Extracted the supernatant with an equal volume of saturated phenol and chloroform∶isoamyl-alcohol (24∶1) in the centrifugation of 10000 g 20 min for twice.
8. Added an equal volume of isopropyl alcohol (about 10 ml) to the upper clear aqueous phase. Then put the centrifuge bottles in the −20°C for 1 hour or overnight.
9. Centrifuged the aqueous phase at 10000 g for 20 min. The cpDNA pellet is washed repeatedly with ethanol (70%, 96%), air-dried, and re-dissolved in 50 µl TE buffer.
10. Treated the cpDNA sample with 2 µl RNAse and visualize the DNA band on a 0.8% agarose gel.
C. sucrose gradient centrifugation [9] (link)
Reagents
Isolation buffer: 0.35 M sorbitol, 50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 0.1% BSA, 0.1% β-mercaptoethanol (v/v);
wash buffer: 0.35 M sorbitol, 50 mM Tris–HCl (pH 8.0), 25 mM EDTA;
Chloroplast isolation
1. Prior to extraction, about 20 g (fresh weight) leaves were collected and kept in dark for 48 to 72 hours at 4°C in order to decrease starch level stored in the leaves.
2. The leaves were cut into pieces (∼1 cm) and homogenized in 400 ml ice-cold isolation buffer for 30 seconds. Filter the homogenate into centrifuge bottles using two layers of Miracloth (Merck) with softly squeezing the cloth.
3. Centrifuged the homogenate (1000 g, 20 min).
4. Resuspended pellet in 7 ml of ice-cold wash buffer using a soft paintbrush.
5. Gently loaded the resuspended pellet onto a step gradient consisting of 18 ml of 52% sucrose, overlayered with 7 ml of 30% sucrose.
6. Centrifuged the step gradients at 25,000 rpm for 60 min at 4°C in a swinging bucket rotor.
7. Removed the chloroplast band from the 30–52% interface using a wide-bore pipette, dilute with 40 ml wash buffer, and centrifuge at 1500 g for 15 min at 4°C.
8. Resuspended the chloroplast pellet with 2 ml wash buffer.
Chloroplast DNA isolation
9. Chloroplast DNA isolation followed steps 6–10 in high salt method.
D. DNAse I treatment [9] (link)
In the DNAse I treatment method, steps were the same with sucrose gradient centrifugation method except the step 9 which was treated with DNAse I. That is, the step 9 in sucrose gradient centrifugation method was substituted with: add 20 µl DNAse I (10 mg/ml) and 250 µl 200 mM MgCl2 to chloroplast solution buffer, incubate at 37°C for 60 min. Then add 1 ml 0.5 M EDTA to terminate the reaction.
A. Modified high salt method (
Reagents
Buffer A (PH 3.8)
1.25 M NaCl, 0.25 M ascorbic acid, 10 mM sodium metabisulfite, 0.0125 M Borax, 50 mM Tris-HCl (pH 8.0), 7 mM EDTA, 1% PVP-40 (w/v), 0.1% BSA (w/v), 1 mM DTT;
Buffer B (PH 8.0)
1.25 M NaCl, 0.0125 M Borax, 1% PVP-40 (w/v), 50 mM Tris-HCl (PH 8.0), 25 mM EDTA, 0.1% BSA (w/v), 1 mM DTT;
Buffer C
100 mM NaCl, 100 mM Tris-HCl (PH 8.0), 50 mM EDTA, 1 mM DTT;
Both BSA and DTT were added just before the start of the experiment.
Chloroplast isolation
All the following steps were carried out at 0°C if not otherwise stated.
1. Prior to extraction, about 20 g (fresh weight) leaves were collected and kept in dark for 48 to 72 hours at 4°C to decrease starch level stored in the leaves.
2. The leaves were nervure-removed, cut into pieces (∼1 cm) and homogenized in 400 ml ice-cold buffer A for 30 seconds. Filter the homogenate into centrifuge bottles using two layers of Miracloth (Merck) by softly squeezing the cloth.
3. Centrifuged the homogenate (200 g, 20 min). The nucleus pellet and cell-wall debris were discarded.
4. Repeated the centrifugation once again. The supernatant included chloroplasts suspended in it.
5. Centrifuged the supernatant at a higher centrifugal force of 3500 g for 20 min, the resulting pellet were chloroplast pellet with some contamination of nuclear DNAs.
6. Added 250 ml Buffer B to the pellet and suspend it gently using a paintbrush to wash the nuclear DNAs attaching to the chloroplast cytomembrane. Then centrifuge with 3500 g for 20 min and discard the supernatant.
7. Re-suspended the pellet with 250 ml Buffer B again and centrifuged (3750 g for 20 min) to gain the purified chloroplasts.
Chloroplast DNA isolation
8. Added 8 ml Buffer C, 1.5 ml 20% SDS, 20 µl β-Me, 30 µl Proteinase K (10 mg/ml) to the purified chloroplast pellet and incubate at 55°C for at least 4 hours or overnight. The chloroplasts would be fully lysed.
9. Put the centrifuge bottles on ice for 5 minutes, add 1.5 ml 5 M KAc (PH 5.2) and continue to freeze for 30 minutes. Then 10000 g centrifuge 15 min, discarding the pellet.
10. Extracted the supernatant with an equal volume of saturated phenol and chloroform∶isoamyl-alcohol (24∶1) in the centrifugation of 10000 g 20 min for twice.
11. Added an equal volume of isopropyl alcohol (about 10 ml) to the upper clear aqueous phase. Then put the centrifuge bottles in the −20°C for 1 hour or overnight.
12. Centrifuged the aqueous phase at 10000 g for 20 min. The cpDNA pellet was washed repeatedly with ethanol (70%, 96%), air-dried, and re-dissolved in 50 µl TE buffer.
13. Treated the cpDNA sample with 2 µl RNAse and visualize the DNA band on a 0.8% agarose gel.
B. high salt method [13] (link)
Reagents
Cold isolation buffer: 1.25 M NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.1% BSA (w/v), 0.1% β-mercaptoethanol (v/v).
Chloroplast isolation
All the following steps were carried out at 0°C if not otherwise stated.
1. Prior to extraction, about 20 g (fresh weight) leaves were collected and kept in dark for 48 to 72 hours at 4°C in order to decrease starch level stored in the leaves.
2. The leaves were cut into pieces (∼1 cm) and homogenized in 100 ml ice-cold Cold isolation buffer for 30 seconds. Filter the homogenate into centrifuge bottles using two layers of Miracloth (Merck) with softly squeezing the cloth.
3. Centrifuged the homogenate (3000 g, 10 min).
4. Resuspended the chloroplast pellet in 30 ml cold isolation buffer, and repellet the chloroplasts (3000 g, 10 min).
5. Resuspended the final chloroplast pellet in 10 ml cold isolation buffer.
Chloroplast DNA isolation
6. Added 1/10 volume of 10% CTAB to lyse the chloroplasts. Incubate at 55°C for 1 to 2 hours.
7. Extracted the supernatant with an equal volume of saturated phenol and chloroform∶isoamyl-alcohol (24∶1) in the centrifugation of 10000 g 20 min for twice.
8. Added an equal volume of isopropyl alcohol (about 10 ml) to the upper clear aqueous phase. Then put the centrifuge bottles in the −20°C for 1 hour or overnight.
9. Centrifuged the aqueous phase at 10000 g for 20 min. The cpDNA pellet is washed repeatedly with ethanol (70%, 96%), air-dried, and re-dissolved in 50 µl TE buffer.
10. Treated the cpDNA sample with 2 µl RNAse and visualize the DNA band on a 0.8% agarose gel.
C. sucrose gradient centrifugation [9] (link)
Reagents
Isolation buffer: 0.35 M sorbitol, 50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 0.1% BSA, 0.1% β-mercaptoethanol (v/v);
wash buffer: 0.35 M sorbitol, 50 mM Tris–HCl (pH 8.0), 25 mM EDTA;
Chloroplast isolation
1. Prior to extraction, about 20 g (fresh weight) leaves were collected and kept in dark for 48 to 72 hours at 4°C in order to decrease starch level stored in the leaves.
2. The leaves were cut into pieces (∼1 cm) and homogenized in 400 ml ice-cold isolation buffer for 30 seconds. Filter the homogenate into centrifuge bottles using two layers of Miracloth (Merck) with softly squeezing the cloth.
3. Centrifuged the homogenate (1000 g, 20 min).
4. Resuspended pellet in 7 ml of ice-cold wash buffer using a soft paintbrush.
5. Gently loaded the resuspended pellet onto a step gradient consisting of 18 ml of 52% sucrose, overlayered with 7 ml of 30% sucrose.
6. Centrifuged the step gradients at 25,000 rpm for 60 min at 4°C in a swinging bucket rotor.
7. Removed the chloroplast band from the 30–52% interface using a wide-bore pipette, dilute with 40 ml wash buffer, and centrifuge at 1500 g for 15 min at 4°C.
8. Resuspended the chloroplast pellet with 2 ml wash buffer.
Chloroplast DNA isolation
9. Chloroplast DNA isolation followed steps 6–10 in high salt method.
D. DNAse I treatment [9] (link)
In the DNAse I treatment method, steps were the same with sucrose gradient centrifugation method except the step 9 which was treated with DNAse I. That is, the step 9 in sucrose gradient centrifugation method was substituted with: add 20 µl DNAse I (10 mg/ml) and 250 µl 200 mM MgCl2 to chloroplast solution buffer, incubate at 37°C for 60 min. Then add 1 ml 0.5 M EDTA to terminate the reaction.
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borax
boric acid
Brain
Buffers
Dissection
Fungus, Filamentous
Ketamine
Light
Mice, Transgenic
Microwaves
Olfactory Bulb
paraform
Perfusion
Phosphates
Saline Solution
Sepharose
sodium borate
sodium borohydride
sodium metaperiodate
Strains
Suction Drainage
Vacuum
Xylazine
Animals were anesthetised with pentobarbital and perfused intracardially with 150 ml of heparinised saline solution followed by 1 ml/g of 4% paraformaldehyde. Spinal cord fragments 2 cm in length (1 cm rostral and 1 cm caudal to the wound epicentre) were immediately dissected, post-fixed for 4 h in the same fixative solution, and then cryoprotected by immersion in 30% sucrose for 48 h and embedded in Neg-50 medium (Richard-Allan Scientific) and stored at −20°C. Parallel serial cryosections (30 µm thick) were then cut, mounted on slides and stored at −20°C.
Cellular ObRb expression was evaluated in different cell types in a set of parallel sections from uninjured (n = 5) and from vehicle and leptin-treated animals sacrificed at 24 h post-injury (seetable S1 ) by dual IHC against adenomatous polyposis coli (APC; oligodendrocytes), NeuN (neurons), GFAP (astrocytes) or OX-42 (microglia/macrophages). Briefly, sections were treated for 1 h at RT with a Blocking Buffer (BB) containing 10% FBS, 0.3% BSA and 0.3% Triton X-100 in (pH 7.4) TBS, followed by incubation overnight at 4°C plus 1 h at RT with a rabbit anti-ObRb (1∶100; Abcam) primary antibody in BB. After washing, the sections were incubated for 1 h at RT with an Alexa 488-linked anti-rabbit secondary antibody (1∶1000; Invitrogen) in BB with 10% rat serum instead of FBS. The sections were then washed again and blocked with BB for 1 h at RT before immunostaining as above but with an Alexa 594-linked anti-mouse antibody and one of the following primary mouse antibodies: APC (1∶100; Calbiochem), NeuN (1∶250; Chemicon), GFAP (1∶1000; Sigma) or OX-42 (1∶500; Wako). Finally, the slides were coverslipped in “Immumount” (Thermo Scientific) and confocal images were collected at RT on a Leica TCS SP5 confocal microscope, with 40×1.25 NA and 63×1.40 NA oil objectives and LAS-AF software (Leica).
To evaluate astroglial and microglia/macrophage reactivity, GFAP and Iba1 IHC were performed on a set of parallel sections from each animal euthanised at the end of the study. Briefly, sections were treated for 10 min with 2% H2O2 in a 70% methanol solution at RT, immersed for 1 h at RT with BB and incubated overnight at 4°C plus 1 h at RT with rabbit anti-GFAP (1∶1000; Dako) or rabbit anti-Iba1 (1∶1000; Wako). After washing, the sections were incubated with a biotinylated anti-rabbit secondary antibody (1∶500; Vector) in BB and then with HRP-linked streptavidin (1∶500; Perkin Elmer) and visualised using the Nova Red Kit (Vector), according to the manufacturer's instructions. The sections were dehydrated in graded ethanol and coverslipped in DPX (Panreac). Images were acquired on a BX61 Motorized Research Microscope (Olympus) and the immunoreactive volume and area were quantified with ImageJ software, either in the total spinal cord volume or at each rostro-caudal spinal cord level analysed (8 sections per animal separated by 600 µm and containing the wound epicentre). The data corresponding to the total immunoreactive volume and area per rostro-caudal level are presented, respectively, as the percentage of total spinal cord volume and spinal cord area per rostro-caudal level.
In each single IHC experiment, processed sections lacking primary antibody were used as controls. For dual immunohistochemistry and to identify putative cross reactivity between the second secondary antibody and the first primary antibody, processed sections lacking the second primary antibody were used as controls.
Myelin preservation was assessed by eriochrome cyanine staining (ECy) in 1 set of parallel sections from each animal at 28 d post-SCI. Briefly, slides were air-dried for 2 h, dehydrated in acetone for 5 min and stained by immersion in 0.2% eriochrome cyanine plus 0.4% iron alum for 30 min. Gray matter (gray) and white (dark blue) matter were differentiated by incubation in 5% iron alum, followed by 1% Borax and 1.25% potassium ferricyanide for 15 min each. Finally, sections were rinsed with tap water, dehydrated with graded ethanol solutions, cleared with xylol and coverslipped with DPX. Myelin preserved areas were analysed by densitometry with ImageJ software. The data corresponding to preserved myelin are presented as the percentage of the spinal cord area in each rostro-caudal level.
Cellular ObRb expression was evaluated in different cell types in a set of parallel sections from uninjured (n = 5) and from vehicle and leptin-treated animals sacrificed at 24 h post-injury (see
To evaluate astroglial and microglia/macrophage reactivity, GFAP and Iba1 IHC were performed on a set of parallel sections from each animal euthanised at the end of the study. Briefly, sections were treated for 10 min with 2% H2O2 in a 70% methanol solution at RT, immersed for 1 h at RT with BB and incubated overnight at 4°C plus 1 h at RT with rabbit anti-GFAP (1∶1000; Dako) or rabbit anti-Iba1 (1∶1000; Wako). After washing, the sections were incubated with a biotinylated anti-rabbit secondary antibody (1∶500; Vector) in BB and then with HRP-linked streptavidin (1∶500; Perkin Elmer) and visualised using the Nova Red Kit (Vector), according to the manufacturer's instructions. The sections were dehydrated in graded ethanol and coverslipped in DPX (Panreac). Images were acquired on a BX61 Motorized Research Microscope (Olympus) and the immunoreactive volume and area were quantified with ImageJ software, either in the total spinal cord volume or at each rostro-caudal spinal cord level analysed (8 sections per animal separated by 600 µm and containing the wound epicentre). The data corresponding to the total immunoreactive volume and area per rostro-caudal level are presented, respectively, as the percentage of total spinal cord volume and spinal cord area per rostro-caudal level.
In each single IHC experiment, processed sections lacking primary antibody were used as controls. For dual immunohistochemistry and to identify putative cross reactivity between the second secondary antibody and the first primary antibody, processed sections lacking the second primary antibody were used as controls.
Myelin preservation was assessed by eriochrome cyanine staining (ECy) in 1 set of parallel sections from each animal at 28 d post-SCI. Briefly, slides were air-dried for 2 h, dehydrated in acetone for 5 min and stained by immersion in 0.2% eriochrome cyanine plus 0.4% iron alum for 30 min. Gray matter (gray) and white (dark blue) matter were differentiated by incubation in 5% iron alum, followed by 1% Borax and 1.25% potassium ferricyanide for 15 min each. Finally, sections were rinsed with tap water, dehydrated with graded ethanol solutions, cleared with xylol and coverslipped with DPX. Myelin preserved areas were analysed by densitometry with ImageJ software. The data corresponding to preserved myelin are presented as the percentage of the spinal cord area in each rostro-caudal level.
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All procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals following protocols approved by the University of California, Davis Institutional Animal Care and Use Committee. Timed-pregnant Sprague Dawley rats were purchased from Charles River Laboratory (Hollister, CA). All animals were housed in clear plastic shoebox cages containing corn cob bedding under constant temperature (22 ± 2 °C) and a 12-h light-dark cycle. Food and water were provided ad libitum. Primary cortical cell cultures were prepared from postnatal day 0 rat pups as previously described [26 (link)]. Neocortices from all pups in the litter were pooled, dissociated, and plated at a density of 650 cells/mm2 on substrates precoated with 0.5 mg/mL of poly-l -lysine (Sigma) in B buffer (3.1 mg/mL boric acid and 4.75 mg/mL borax, Sigma) for 4 h at 37 °C and 5% CO2 then washed with sterile deionized water and covered with plating medium. Primary cortical cells were plated in plating medium and allowed to adhere for 4 h before the medium was changed to the co- or tri-culture medium. Half-media changes were performed at DIV 3, 7, and 10 with the respective media types.
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Animals
Animals, Laboratory
borax
boric acid
Buffers
Cells
Culture Media
Food
Institutional Animal Care and Use Committees
Kidney Cortex
Lysine
Maize
Neocortex
Poly-5
Primary Cell Culture
Rats, Sprague-Dawley
Rivers
Sterility, Reproductive
Most recents protocols related to «Borax»
For the boric acid standard solution preparation, 1 mg, 1.5 mg, 2.5 mg, 3.5 mg, 5 mg, 7.5 mg, and 10 mg of weighed boric acid were dissolved in 0.5 mL of D2O.
For the borax standard solution preparation, 1.5 mg, 2.5 mg, 3.5 mg, 5 mg, 7.5 mg, and 10 mg of weighed borax were dissolved in 0.5 mL of D2O. All relevant standard solutions in the experiments of this paper were prepared without using pH buffer solutions.
11B NMR solutions of borax were prepared at various pHs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. A weighed amount of borax was dissolved in a D2O solution, After borax had been completely dissolved, the pH of the solution was adjusted with NaOH or acetic acid. The pH detection of borax aqueous solution was carried out using pH precision test strips. For the 11B NMR study of borax solutions at various temperatures, 10 mg of borax was dissolved in 0.5 mL of D2O. The temperature control switch of the NMR spectrometer was turned on and an 11B NMR experiment was conducted after stabilizing at the target temperature.
For the borax standard solution preparation, 1.5 mg, 2.5 mg, 3.5 mg, 5 mg, 7.5 mg, and 10 mg of weighed borax were dissolved in 0.5 mL of D2O. All relevant standard solutions in the experiments of this paper were prepared without using pH buffer solutions.
11B NMR solutions of borax were prepared at various pHs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. A weighed amount of borax was dissolved in a D2O solution, After borax had been completely dissolved, the pH of the solution was adjusted with NaOH or acetic acid. The pH detection of borax aqueous solution was carried out using pH precision test strips. For the 11B NMR study of borax solutions at various temperatures, 10 mg of borax was dissolved in 0.5 mL of D2O. The temperature control switch of the NMR spectrometer was turned on and an 11B NMR experiment was conducted after stabilizing at the target temperature.
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SA-CS polymer and PVA were separately dissolved in phosphate-buffered saline (5 mL, PBS) at a pH of 7.2 at 37 o C to prepare a mixed solution containing SA-CS (3.5% w/v) and PVA (3.5% w/v). Then, 0.000 g, 0.001g, 0.015g, and 0.025g Cur-PF127 were respectively added to the aforementioned mixed solution to make the concentration of Cur-PF127 at 0%, 1%, 3%, and 5% (w/v). Subsequently, 900 μL 4 % wt of borax was introduced to the mixture solution and vortexed to obtain a series of composite hydrogels referred to as Gel 1 to Gel 4, respectively.
borax (HT1002, Sigma‐Aldrich) was solubilized in either 0.1% dimethyl sulfoxide (DMSO; 20688, Thermo Scientific) or complete cell culture medium at 25°C. Subsequently, the stock solution (0.4 M) was further diluted to attain the necessary concentrations for various assays. On the day of the experiment, the stock solution was filtered and prepared fresh. Before treatment, HL7702 and HepG2 cells were seeded in 96‐well plates and incubated in culture medium for 24 h until approximately 80% confluency. Subsequently, the cells were treated with borax concentrations ranging from 0.5 to 64 mM for up to 72 h. Following, measurements were made at 570 nm using the cell viability analysis kit (TOX1‐1KT; Sigma‐Aldrich) based on 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) analysis, in accordance with the manufacturer's instructions. The untreated control group's cell viability was designated as 100%, and the viability of cells subjected to borax treatment was computed relative to this control group. The cell viability curve was constructed using the calculated percentage values of cell viability for each concentration of borax, and the concentrations corresponding to 25% (IC25) and 50% (IC50) inhibition were ascertained from this curve.
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To assess the impact of borax on the proliferation of HepG2 and HL‐7702 cells (1 × 105), seeded in 12‐well plates, we employed a commercially available 5‐bromo‐2′‐deoxyuridine (BrdU) kit (2750; Sigma‐Aldrich). HepG2 and HL‐7702 cells were treated with borax concentrations determined according to MTT results for 24 h. The proliferation rate of cells was then analysed colorimetrically based on BrdU incorporation assay, which detects DNA synthesis during cellular replication, following the manufacturer's instructions. Absorbance values were analysed at 450 nm using a microplate reader (BioTek).
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To assess cell viability, the Enhanced Cell Counting Kit 8 (WST‐8/CCK8, E‐CK‐A362, Elabscience) was utilized. WST‐8 is a compound similar to MTT that can be reduced to orange formazan by mitochondrial dehydrogenases in the presence of an electron coupling reagent. The amount of formazan produced is directly proportional to the number of viable cells. In the experiment, the cells were seeded into 96‐well plates at a density of 5 × 103 cells per well. Once the cells adhered to the bottom of the plates, varying concentrations of borax (ranging from 0 to 800 μM) were added to the wells. The concentration range for borax was determined through preliminary treatments. After specific treatment durations (24, 48 or 72 h), the cell culture media were removed from the wells. The absorbance at 450 nm was measured to indirectly calculate the amount of viable cells. The average viability of untreated cells was considered as 100%, and the viability percentages of the cells treated withs borax were calculated proportionally. The following formula was used to calculate the viability percentages: Cell viability: (ODtreated cells−ODblank)/(ODcontrol cells−ODblank) × 100.
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Top products related to «Borax»
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Borax is a naturally occurring mineral compound that is commonly used as a laboratory reagent. It consists of sodium, boron, and oxygen, and its primary function is to serve as a buffer and source of borate ions in various laboratory applications.
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Sodium hydroxide is a chemical compound with the formula NaOH. It is a white, odorless, crystalline solid that is highly soluble in water and is a strong base. It is commonly used in various laboratory applications as a reagent.
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Boric acid is a chemical compound with the formula H3BO3. It is a white, crystalline solid that is soluble in water. Boric acid is commonly used as a laboratory reagent and is a component in various products, including eye drops, antiseptics, and insecticides.
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Hydrochloric acid is a commonly used laboratory reagent. It is a clear, colorless, and highly corrosive liquid with a pungent odor. Hydrochloric acid is an aqueous solution of hydrogen chloride gas.
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Epon 812 is an epoxy resin that is commonly used as an embedding medium for electron microscopy sample preparation. It is a low-viscosity, hard, and durable resin that provides a stable support for delicate biological specimens during sectioning and analysis.
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Technovit 7100 is a two-component resin system designed for embedding and preparing samples for microscopic analysis. It is a cold-curing resin that allows for the preservation of the original structure and morphology of the embedded samples.
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The Olympus BX60 is a light microscope designed for general laboratory use. It features a sturdy, ergonomic design and provides clear, high-quality images. The BX60 is capable of bright-field, phase contrast, and simple polarized light microscopy techniques.
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Toluidine blue is a phenazine dye used in various laboratory applications. It is a metachromatic stain that can be used to differentiate different types of tissues and cellular components.
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Methanol is a clear, colorless, and flammable liquid that is widely used in various industrial and laboratory applications. It serves as a solvent, fuel, and chemical intermediate. Methanol has a simple chemical formula of CH3OH and a boiling point of 64.7°C. It is a versatile compound that is widely used in the production of other chemicals, as well as in the fuel industry.
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BrdU is a synthetic nucleoside that is an analog of the DNA base thymidine. It can be incorporated into the newly synthesized DNA of replicating cells, substituting for thymidine during the DNA synthesis phase of the cell cycle.
More about "Borax"
Borax, also known as sodium borate or sodium tetraborate, is a naturally occurring mineral compound with a wide range of industrial and research applications.
This versatile chemical is commonly used as a flux in metallurgy, a preservative, an insecticide, and a cleaning agent.
In scientific research, borax plays a crucial role in various applications, such as histological staining, electron microscopy sample preparation, and materials science.
Closely related to borax are other boron-based compounds like boric acid and sodium hydroxide, which are also used extensively in industry and research.
Boric acid, for instance, is a common ingredient in eye drops and ointments, while sodium hydroxide, also known as caustic soda, is a key component in the production of soaps, detergents, and other cleaning products.
The PubCompare.ai platform is an AI-driven tool that can optimize your borax research by helping you locate the best protocols and products.
By comparing data from scientific literature, preprints, and patents, this platform enhances the reproducibility and accuracy of your borax studies, allowing you to make more informed decisions and achieve better results.
In addition to borax, PubCompare.ai can also assist with research involving other common laboratory reagents and materials, such as Epon 812, Technovit 7100, and the BX60 light microscope.
These tools are essential for various applications, including tissue embedding, sectioning, and staining, which are often used in borax-related research.
Incorporating synonyms and abbreviations, such as sodium borate (Na2B4O7) and BrdU (bromodeoxyuridine), can further enrich the content and make it more accessible to researchers.
By addressing key subtopics and related terms, this information-packed text provides a comprehensive overview of the multifaceted world of borax and its applications in industry and scientific research.
This versatile chemical is commonly used as a flux in metallurgy, a preservative, an insecticide, and a cleaning agent.
In scientific research, borax plays a crucial role in various applications, such as histological staining, electron microscopy sample preparation, and materials science.
Closely related to borax are other boron-based compounds like boric acid and sodium hydroxide, which are also used extensively in industry and research.
Boric acid, for instance, is a common ingredient in eye drops and ointments, while sodium hydroxide, also known as caustic soda, is a key component in the production of soaps, detergents, and other cleaning products.
The PubCompare.ai platform is an AI-driven tool that can optimize your borax research by helping you locate the best protocols and products.
By comparing data from scientific literature, preprints, and patents, this platform enhances the reproducibility and accuracy of your borax studies, allowing you to make more informed decisions and achieve better results.
In addition to borax, PubCompare.ai can also assist with research involving other common laboratory reagents and materials, such as Epon 812, Technovit 7100, and the BX60 light microscope.
These tools are essential for various applications, including tissue embedding, sectioning, and staining, which are often used in borax-related research.
Incorporating synonyms and abbreviations, such as sodium borate (Na2B4O7) and BrdU (bromodeoxyuridine), can further enrich the content and make it more accessible to researchers.
By addressing key subtopics and related terms, this information-packed text provides a comprehensive overview of the multifaceted world of borax and its applications in industry and scientific research.