We dissolved probiotics purchased from local pharmacies in sterile 1× phosphate-buffered saline (PBS; Fisher Bioreagents: BP3991) in the anaerobic chamber. The dissolved slurry was streaked onto agar plates and incubated anaerobically at 37°C for 24 h. Both PBS and medium were pre-reduced in an anaerobic chamber for at least 24 h before use. We isolated Lactobacillaceae using Mega Medium and Bifidobacteriaceae using Bifidobacterium selective iodoacetate mupirocin medium according to a previously published method (Table S1 ) (55 (link)).
Bp3991
Manufactured by Thermo Fisher Scientific
The BP3991 is a laboratory centrifuge designed for general-purpose applications. It features a fixed-angle rotor that can accommodate various sample tubes and microplates. The centrifuge provides adjustable speed control and a timer function to meet the needs of various sample preparation and separation workflows.
Automatically generated - may contain errors
Lab products found in correlation
Polyethylenimine (pei), by Merck Group (2 mentions)
G6257, by Merck Group (2 mentions)
Oasis hlb column, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
Type 1 collagen, by BD (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Dsc20010 1000, by Merck Group (1 mentions)
Bead mill 24, by Biospec (1 mentions)
Thermomixer f1, by Eppendorf (1 mentions)
Zirconia beads, by Biospec (1 mentions)
P36931, by Thermo Fisher Scientific (1 mentions)
Colorfrost plus microscope slides, by Thermo Fisher Scientific (1 mentions)
Hm550, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Decorin, by Merck Group (1 mentions)
Dfc365fx, by Leica (1 mentions)
Dmi4000b, by Leica (1 mentions)
P35g 0 14 c, by MatTek (1 mentions)
T200 evaluation software, by Cytiva (1 mentions)
Biacore t200, by Cytiva (1 mentions)
14 protocols using bp3991
1
Anaerobic Isolation of Probiotics
2
Fabricating Collagen Vessel Structures
3
Protein Extraction and Digestion Protocol
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Cells were
washed twice with cold phosphate-buffered saline (PBS) (Fisher Scientific,
BP399-1) and pelleted by centrifugation (1000 rpm, 5 min). Lysis buffer
containing 8 M urea (Fisher Scientific, BP169) and 50 mM Tris (Thermofisher
Scientific) pH 8.2 was added to the cell pellets. Cells were mechanically
lysed with 2 × 10 s sonication bursts. Lysates were centrifuged
at 14,000g for 10 min, and protein concentration
of the clear lysate was determined by Bradford assay. Samples were
diluted to 1 M urea and 50 mM Tris, and Lys-C (Wako Pure Chemical
Corp.) (enzyme/protein ratio 1:100) digestion was performed for 3
h at 37 °C. Trypsin (Promega) digestion (enzyme/protein ratio
1:50) was performed overnight at 37 °C. Samples were desalted
with OASIS HLB columns (Waters) and dried in a speed-vac. Samples
were reconstituted in formic acid 4% (EMD Millipore Corporation).
washed twice with cold phosphate-buffered saline (PBS) (Fisher Scientific,
BP399-1) and pelleted by centrifugation (1000 rpm, 5 min). Lysis buffer
containing 8 M urea (Fisher Scientific, BP169) and 50 mM Tris (Thermofisher
Scientific) pH 8.2 was added to the cell pellets. Cells were mechanically
lysed with 2 × 10 s sonication bursts. Lysates were centrifuged
at 14,000g for 10 min, and protein concentration
of the clear lysate was determined by Bradford assay. Samples were
diluted to 1 M urea and 50 mM Tris, and Lys-C (Wako Pure Chemical
Corp.) (enzyme/protein ratio 1:100) digestion was performed for 3
h at 37 °C. Trypsin (Promega) digestion (enzyme/protein ratio
1:50) was performed overnight at 37 °C. Samples were desalted
with OASIS HLB columns (Waters) and dried in a speed-vac. Samples
were reconstituted in formic acid 4% (EMD Millipore Corporation).
4
Fabrication of Collagen Hydrogel Lumen
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LumeNEXT microdevices [22 (link)] (fabrication details in SI) were treated 10 min with 1% poly(ethyleneimine) (Sigma-Aldrich, 03880) in water and with 0∙1% glutaraldehyde (Sigma-Aldrich, G6257) in water for 30 min to enhance hydrogel attachment to the PDMS. Devices were washed at least three times with water and thoroughly dried before hydrogel injection. Briefly, Type I rat tail collagen hydrogels were prepared at 6 mg/ml in ice to prevent premature gelation. Using a chilled tip, a mixture of 80 μl of collagen type I (10 mg/ml, BD Biosciences, 354,249); 3∙5 μl NaOH 0.5 M (Fisher Scientific, S318); 10 μl PBS 10× (Fisher Scientific, BP3991) and 34 μl of PBS was prepared and incubated on ice for 20 min. The pH was then checked with colorimetric strips (Capitol Scientific, PH1170–7). Provided the pH of the mixture be approximately 7∙4, the mixture was injected into the LumeNext device, and polymerized for 5–10 min at room temperature, followed by 20 min at 37 °C. Afterward, the PDMS rods were pulled out of the polymerized collagen gel from the output port resulting in a lumen structure in the collagen gel.
5
Optimizing Hydrogel Adhesion in Microdevice
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To achieve maximum hydrogel adhesion to the device chamber, a 2-step coating of 2% poly(ethyleneimine) (PEI, Sigma-Aldrich, 03880, St Louis, MO, USA) diluted in deionized DI water for 10 min was loaded into the side ports. The PEI solution was aspirated, and 0.4% glutaraldehyde (GA, Sigma-Aldrich, G6257) diluted in deionized (DI) water was loaded into the side ports and incubated at room temperature for 30 min. During the GA incubation, the collagen solution was prepared on ice (refer to Section 4.3.2 ). After the 30-minute GA incubation, the microdevices were washed 3 times with sterile DI water to remove any GA excess. At this point, devices were ready to be loaded with the collagen solution. To minimize evaporation, sacrificial phosphate-buffered saline (PBS, Fisher scientific, BP3991) was added around the side of the MatTek dish.
6
Tissue Homogenization and Digestion Protocol
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Obex samples were homogenized using bead beater (1 min, 4 m/s; Fisherbrand Bead Mill 24) with 0.7 mm diameter zirconia beads (BioSpec cat. no. 11079107zx) in 1× PBS (Fisher Scientific BP3991) to produce a 10% wt/v suspension. Samples were centrifuged (2 min, 3,000g), and the supernatants were collected. The supernatants were centrifuged again (3 min, 3,000g), aliquoted into 10 μL samples, and frozen at –80°C.
All other tissues were digested prior to analysis using the following protocol. A 10% wt/v solution of tissue was prepared in a solution of 1× PBS, 2 mM CaCl2 (Dot Scientific DSC20010-1000), and 0.25% wt/v collagenase A (Sigma-Aldrich 10103586001) [19 (link), 22 (link)]. Samples were homogenized with a bead beater and zirconia beads as above. The samples were then shaken with a thermomixer (24 h, 45°C; Eppendorf ThermoMixer F1.5). After agitation and incubation, the samples were centrifuged (2 minutes, 3,000g), and the supernatants were retained. The supernatants were centrifuged again (3 min, 3,000g) to remove any small particulate matter, aliquoted, and frozen at –80°C.
All other tissues were digested prior to analysis using the following protocol. A 10% wt/v solution of tissue was prepared in a solution of 1× PBS, 2 mM CaCl2 (Dot Scientific DSC20010-1000), and 0.25% wt/v collagenase A (Sigma-Aldrich 10103586001) [19 (link), 22 (link)]. Samples were homogenized with a bead beater and zirconia beads as above. The samples were then shaken with a thermomixer (24 h, 45°C; Eppendorf ThermoMixer F1.5). After agitation and incubation, the samples were centrifuged (2 minutes, 3,000g), and the supernatants were retained. The supernatants were centrifuged again (3 min, 3,000g) to remove any small particulate matter, aliquoted, and frozen at –80°C.
7
Quantifying Baboon Brain Nuclei
8
Cell Viability Assay by Flow Cytometry
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Cells were plated on 6 well plates and allowed to adhere for a 24‐hour period. Cells were then treated. Each 6‐well plate had a DMSO vehicle control that was set to 100% viability with which the other treatments were normalized to; each plate also had a cell‐free control. After 48 h, cells were trypsinized and received 500 μl of 70% ethanol dropwise while being vortexed. Cells were then refrigerated for 3–4 h at 4°C. Next cells were centrifuged and washed with 1× PBS (Fisher Bioreagents, BP399‐1). After adding 200 μl of Propidium iodide, made at 50 μg/ml in 1× PBS (Caymen Chem, 14 289), and 1.3 ml of 1× PBS, cells were vortexed. Cells were then run and analyzed by the Accuri C6+ Flow Cytometer (BD Biosciences, Mississauga, ON, Canada).
9
Bovine Collagen Fiber Alignment
10
Fluorescence Microscopy of Adherent MEFs
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The MEFs were applied onto glow-discharged EM grids with a silicon-oxide support film (R1/4, Au mesh; Quantifoil, Jena, Germany). After incubation, the cells were fixed in a 4% paraformaldehyde solution (Sigma-Aldrich, 16005), and washed 3 times with 1x PBS (Fisher Scientific, BP399-1). The EM grids were transferred to a 35x14 mm glass-bottom cell culture dish (MatTek, P35G-0-14-C) for fluorescence microscopy analysis of the FAs. Adherent MEFs were imaged using an automated inverted microscope (DMI4000 B, Leica Microsystems, Wetzlar, Germany) equipped with a fluorescence lamp and a monochromatic digital camera (DFC365 FX, Leica Microsystems). Overview images of the EM grids were acquired using a 10x dry objective (HCX PL Fluotar 10x/0.30, Leica Microsystems). All images of individual cells (Figure 2 A) were acquired in phase contrast and fluorescence mode using a 63x oil objective (HCX PL APO 63x/1.40-0.6, Leica Microsystems).
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