Colon was removed and placed in cold Ca2+, Mg2+-free Hank’s balanced salt solution (HBSS; Gibco). After removal of the mesentery, the colon was opened longitudinally, thoroughly washed in HBSS and cut into small pieces. The dissected mucosa was incubated with HBSS containing 1 mM dithiothreitol (Sigma-Aldrich) and 5 mM EDTA (Quality biological) for 30 min at 37°C to remove the epithelial layer. The pieces of intestine were washed and placed in HBSS containing 1.5% FBS, 200 U/mL collagenase Type 3 and 0.01 mg/mL DNase (all Worthington Biochemical Corporation) for 1 h at 37°C. The digested tissues were washed, resuspended in 40% Percoll (GE Healthcare) and overlaid on a 75% Percoll fraction. Percoll gradient separation was performed by centrifugation at 700 × g for 20 min at room temperature. Mononuclear cells were collected at the interphase, washed, and resuspended in staining buffer containing PBS, 0.5% BSA, 2 mM EDTA for flow cytometry or RPMI-1640 medium (Sigma-Aldrich) containing 10% FBS.
Ethylene diamine tetraacetic acid (edta)
Manufactured by Quality Biological
Sourced in United States
EDTA is an anticoagulant that is commonly used in laboratory settings. It functions by chelating calcium ions, which are necessary for the blood clotting process. This helps prevent the formation of blood clots in collected samples, allowing for accurate testing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 3, by Worthington (4 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (4 mentions)
Percoll, by GE Healthcare (4 mentions)
Dithiothreitol (dtt), by Merck Group (4 mentions)
Hanks balanced salt solution (hbss), by Worthington (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Collagenase 4, by Merck Group (3 mentions)
Phosphatase inhibitors cocktail, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Radioimmunoprecipitation assay (ripa), by Boston BioProducts (3 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Dnase, by Worthington (2 mentions)
Percoll solution, by GE Healthcare (2 mentions)
Dnase 1, by Worthington (2 mentions)
Hanks balanced salt solution (hbss), by Quality Biological (2 mentions)
Trypsin, by Quality Biological (2 mentions)
Penicillin, by Quality Biological (2 mentions)
Streptomycin, by Quality Biological (2 mentions)
16 protocols using ethylene diamine tetraacetic acid (edta)
1
Isolation of Intestinal Mononuclear Cells
2
Isolation of Lamina Propria Mononuclear Cells
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Lamina propria mononuclear cells (LPMCs) were isolated from SI specimens using a modification of a previously described protocol5 (link),32 (link). Briefly, the dissected mucosal tissue was incubated in calcium and magnesium-free Hank’s balanced salt solution (HBSS) (Life Technologies, Carlsbad, CA) containing 1.5% heat-inactivated fetal bovine serum (FBS) (Life Technologies) and 1 mM dithiothreitol (Sigma-Aldrich) to remove mucus. Epithelial cells were removed by incubation in HBSS containing 1 mM EDTA (Quality Biological, Gaithersburg, MD) at 37 °C, repeated 2 times (first incubation; 10 min, second incubation; 30 min). The tissues were then collected and incubated, with agitation, in HBSS containing 400 U ml−1 of type 3 collagenase and 0.01 mg ml−1 of DNase I (Worthington Biochemical, Lakewood, NJ) for 90 min at 37 °C. The insoluble fraction was pelleted, re-suspended in a 40% Percoll solution (GE Healthcare Life Sciences, Pittsburgh, PA), layered on top of a 75% Percoll solution and centrifuged at 2,000 r.p.m. for 20 min at room temperature. Viable LPMCs were recovered from the discontinuous gradient interface.
3
Flow Cytometric Analysis of BMMO Apoptosis
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In sterile 24‐well plates, Day 8 BMMØs were seeded at 1 × 105 cells/well in complete BMMØ media incubated at 37°C and 5% CO2 overnight, as previously described.
17 (link) Subsequently, the media was replaced with BMMØ complete media supplemented with 30 μM SAHA‐OH, 300 μg/mL iNP, 300 μg/mL iNP‐SAHALow, or 300 μg/mL iNP‐SAHAHigh. Three hours later, PBS washing allowed for the removal of excess iNP or iNP‐SAHA followed by replacing with complete BMMØ media containing 100 ng/mL LPS. The cells were lifted using Versene after 48 h and washed with MACS buffer (cold PBS supplemented with 1% FBS and 0.4% 0.5 M EDTA [Quality Biological, Gaithersburg, MD]). FcR blocking (purified anti‐mouse CD16/32 Ab [#101301] [BioLegend, San Diego, CA]) was performed prior to FITC‐annexin V (AV) and propidium iodide (PI) staining [#640914) (BioLegend, San Diego, CA) for apoptosis and cell death, according to the manufacturer's instructions. Becton Dickinson LSR II or Becton Dickinson Canto II flow cytometer was used to analyze the flow cytometry samples. FCS Express 7 Flow Cytometry De Novo Software (De Novo, Glendale, CA) was used for flow data processing.
17 (link) Subsequently, the media was replaced with BMMØ complete media supplemented with 30 μM SAHA‐OH, 300 μg/mL iNP, 300 μg/mL iNP‐SAHALow, or 300 μg/mL iNP‐SAHAHigh. Three hours later, PBS washing allowed for the removal of excess iNP or iNP‐SAHA followed by replacing with complete BMMØ media containing 100 ng/mL LPS. The cells were lifted using Versene after 48 h and washed with MACS buffer (cold PBS supplemented with 1% FBS and 0.4% 0.5 M EDTA [Quality Biological, Gaithersburg, MD]). FcR blocking (purified anti‐mouse CD16/32 Ab [#101301] [BioLegend, San Diego, CA]) was performed prior to FITC‐annexin V (AV) and propidium iodide (PI) staining [#640914) (BioLegend, San Diego, CA) for apoptosis and cell death, according to the manufacturer's instructions. Becton Dickinson LSR II or Becton Dickinson Canto II flow cytometer was used to analyze the flow cytometry samples. FCS Express 7 Flow Cytometry De Novo Software (De Novo, Glendale, CA) was used for flow data processing.
4
Isolation of Placental Leukocytes
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Single cells from explants or in vivo placentas were prepared by manual and enzymatic (collagenase D, Roche, Indianapolis, IN, USA) digestion followed by passing through a 70‐μm nylon mesh (BD Falcon cell strainer). Red blood cell lysis (ACK lysing buffer, Thermo Fisher Scientific) and total cell counts were performed followed by staining of a surface marker for leukocytes and anti-mouse CD45 (clone 30-F11, Thermo Fisher Scientific). The staining was incubated in fluorescence-activated cell sorting (FACS) buffer with 1 mM EDTA (Quality Biological, Gaithersburg, MD, USA) for 30 min at 4°C in the dark. OneComp eBeads (eBioscience) were used for compensation. Data were acquired on an Attune™ NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific) and analyzed with FlowJo version 10.1 (FlowJo, LLC, Ashland, OR, USA). Debris and doublets were excluded by sequential gating on forward scatter height versus forward scatter area followed by gating on CD45+ leukocytes.
5
Whole-mount Immunolabeling of Cochlear Turns
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Whole-mount immunostaining and imaging was performed as previously described (Vyas et al., 2019 (link)). Briefly, grossly dissected inner ears were perfused with 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in 1× PBS (Quality Biological Inc.) through the round window, then postfixed for 30 min on a 3D-Rotator. Mature inner ears were washed (3 × 30 min) in PBS, then decalcified in 125 mm EDTA (Quality Biological Inc.) in PBS for 2 h before dissection into turns of the organ of Corti. Postnatal day 7–10 (P7–10) inner ears were not decalcified before washing in PBS (3 × 30 min) and dissection into turns of the organ of Corti. Cochlear turns were immunolabeled with Goat anti-GFP (Sicgen, catalog #A32814, RRID:AB_2333099 ) to label GCaMP6f protein. Donkey anti-goat Alexa Fluor 488 (Invitrogen, catalog #AB0020-200, RRID:AB_2762838 ) was used along with Alexa Fluor 647-conjugated phalloidin (Thermofisher Scientific, catalog #A22287, RRID:AB_2620155 ) and 4'6-diamidine-2'-phenylindole dihydrochloride (DAPI; Roche Molecular Systems) for GCaMP6f, hair cells, and nuclei, respectively (Fig. 1 ). Cochlear turns were mounted using ProLong Fade Gold Antifade Mountant (Thermofisher Scientific, catalog #P36930) and imaged on a 700 LSM Zeiss confocal microscope.
6
Developmental Histology of Mouse Craniofacial Tissues
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E12.5 to E18.5 embryos, and P0/P1, P7, P14 (n = 3) mice pups were used for morphological observation using hematoxylin and eosin (HE) staining. Whole embryo/pup heads were fixed in 10% neutral buffered formalin (NBF) (Azer Scientific, Cat No. NAF-4-G) for 24 h and dehydrated and processed, paraffin-embedded, and serially sectioned (4.5 μm) in the coronal (molar) and sagittal (incisor) plane. Stages at P0 or later were decalcified using EDTA (0.5M, pH 8.0, Quality Biological, Cat No. 351-027-101). The sections were deparaffinized, rehydrated, stained with H&E, and imaged on an AxioScan.Z1 slide scanner (Zeiss).
7
Isolation of Tumor Cells from Primary Tumors
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We modified and used previously reported methods to isolate tumor cells from primary tumors55 (link),56 . Briefly, MMTV-PyMT tumors from 15-week-old MMTV-PyMT mice or MVT-1 tumors from mice injected with MVT-1 cells at 5 weeks were collected. The tumor tissue was digested in PBS buffer containing 0.5% BSA (Sigma), 2 mM EDTA (Quality Biological), collagenase II (Sigma), collagenase IV (Sigma) and DNase I (Sigma) for 15 min at 37 °C. After incubation, the cells were filtered with 100 μm cell strainer (Corning) and were added with ACK lysing buffer (Quality Biological) to remove RBCs and plated into 10 cm dish. After 16 h, the cells were replated as needed for further experiments.
8
Chinese Hamster Ovary Cell Culture Protocol
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Chinese hamster ovary (CHO) E77.4 cells79 (link) from ATCC were cultured in RPMI-1640 medium (Quality Biological 112-025-101) containing 10% heat-inactivated fetal bovine serum (Gibco™ 10082147), 2mM L-Glutamine (Quality Biological 118-084-721), and 100 U/mL Penicillin with 100 µg/mL Streptomycin (Quality Biological 120-095-721). Passages were performed using 0.05% Trypsin-0.1% EDTA (Quality Biological 118-087-721).
9
Isolation of Intestinal Mononuclear Cells
10
Isolation and Culture of Immune Cells
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Collagenase IV (#C2139-1G) and IGEPAL CA-630 (#I8896) were purchased from Sigma. HBSS (#04-315Q) was purchased from Lonza. TGF-beta (#7666-MB) and MCSF (#216-MC-005) was purchased from R&D systems. DMEM/F12 (11320-033), RPMI 1640 (#11835-030), MEM NEAA (#11140-050), HEPES (#15630-080), HBSS with CaCl (#14025-092), Pen Strep (#15070-063), and PBS (#10010-023) were purchased from Gibco. EDTA (#351-027-721) was obtained from Quality Biological. Percoll (#17-0891-01) was purchased from GE Healthcare. LPS (ALX-581-008-L001) was purchased from Enzo. Trizol (#15596018) was purchased from Ambion. Vismodegib (#879085-55-9) was purchased from LC labs. Hh-Ag1.5 (#C4412) was obtained from collagen technology.
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