DRGs were dissected from either embryonic day 13.5 (E13.5) or postnatal day 4 (P4) mice. Embryonic DRG neurons were digested in collagenase A (1 mg/mL, Roche) for 15 min, followed by trypLE (0.05%, Life Technologies) for 5 min at 37°C and postnatal DRG neurons were digested in collagenase A (1 mg/mL) for 25 min, followed by trypLE (0.05%) for 7 min at 37°C. Enzyme-digested embryonic and postnatal DRGs were washed three times with MEM containing 10% fetal bovine serum and dissociated in cultured medium containing NGF (25 ng/mL, Alomone labs). Dissociated neurons were then plated on glass coverslips coated with a mixture of 100 μg/mL poly-D-lysine and 10 μg/mL laminin (Gibco) and grown in MEM containing NGF (25 ng/mL, Alomone labs) and antibiotics (20 μM 5-fluoro-2-deoxyuridine and 20 μM uridine). 5 hr after plating, neurons were treated with epothilone B (from 1 pM to 1 μM in Figures 2 and 3 and 10 pM or 1 nM in Figure 4 ) overnight (11 hr treatment for Figure 2(a) ; either 11 hr or 17–19 hr treatment for Figures 2(b) and 2(c) ; and 19 hr treatment for Figure 3 ). Neurons were then fixed and analyzed for viability, axon growth, and microtubule structure. For nocodazole treatment, neurons were cultured overnight to allow axon growth and then exposed to either 50 nM for 1.5 hr (Figure 5 ) or 1 μM for 30 min (Figure 6 ), right before fixation.
Collagenase a
Manufactured by Thermo Fisher Scientific
Sourced in United States
Collagenase A is an enzyme used in various laboratory applications. It is a mixture of proteolytic enzymes that breaks down collagen, a major structural component of the extracellular matrix. Collagenase A is commonly used in cell isolation and tissue dissociation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dispase 2, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Mesenpro rs basal media, by Thermo Fisher Scientific (2 mentions)
Mesenpro rs growth supplement, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Collagenase a, by Roche (1 mentions)
Nerve growth factor (ngf), by Alomone (1 mentions)
Facsaria fusion, by BD (1 mentions)
C5894, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions)
Murine epidermal growth factor, by Merck Group (1 mentions)
Nutlin, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
18 protocols using collagenase a
1
Embryonic and Postnatal Dorsal Root Ganglion Neuron Culture
2
Isolation of Bone Marrow Endothelial Cells
3
Isolation of Adipose-Derived Stem Cells from Rats
4
Detecting α-SMA and Cytokines in Lungs
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To detect the production of α‐SMA in the lung, lung tissues were incubated with 1 mg/mL Collagenase A (#17100017; Gibco, Gaithersburg, MD, USA), 1500 kU/mL DNase I (#D5025‐150KU; Sigma‐Aldrich Chemical Co., St. Louis, MO, USA) and CaCl2 (0.025M) at 37℃ for 4 hours to prepare single cells suspension, as previously reported.14 For detecting the cytokines levels in the mediastinal lymph node (mLN) and spleen, lymph nodes and spleen were isolated and grinded with ground glasses, and cells were prepared for further detection by flow cytometry.
5
Tumor Organoid Establishment from Mouse Models
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Firrm−/−;Trp53−/− and Trp53−/− organoids were derived from cryopreserved mammary tumor pieces and processed as previously described by Duarte et al. (47 (link)). Briefly, tumor material was dissected and digested using collagenase A (2 mg/ml; Gibco) in advanced DMEM/F12, washed in growth medium (advanced DMEM/F12, 10 mM Hepes, GlutaMAX, and penicillin-streptomycin) and filtered through a cell strainer. Organoids were then cultured in growth medium further supplemented with B27 (Gibco), 125 μM N-acetyl-l -cysteine (Sigma-Aldrich), and murine epidermal growth factor (50 ng/ml; Sigma-Aldrich). Cells were embedded in 1:1 Culturex Reduced Growth Factor Basement Membrane Extract (BME) Type 2 (Trevigen) mixed with medium and maintained at 37°C and 5% CO2. To establish organoid cultures, cells were supplemented with 5 μM nutlin (Sigma-Aldrich) for the initial 3 weeks of culture.
6
Multisite Immune Cell Profiling
7
Isolation and Characterization of ADMSCs
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ADMSCs were isolated from inguinal fat pad of Sprague–Dawley rats (Slack Experimental Animal Company, Shanghai, China) based on a published method.20 (link) Briefly, the lipoaspirate was minced and digested with 1% collagenase A (Gibco, Carlsbad, CA) and washed with phosphate-buffered saline (PBS). After filtration and centrifugation, cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% fetal calf serum (FCS; Gibco) and cultured in a humidified 5% CO2 atmosphere at 37°C; the cells in passages 3 and 4 were identified and cultured for future experiments.
The collected ADMSCs were then observed by microscope and analyzed by flow cytometry. About 5 × 105 ADMSCs were resuspended in 1 mL of PBS and incubated at 4°C for 30 minutes with the following antirat antibodies (all from BD Biosciences, San Jose, CA): phycoerythrin-conjugated CD29, CD44, and D-related human leukocyte antigen (HLA-DR); fluorescein isothiocyanate–conjugated CD31, CD45, and CD105. Phycoerythrin-conjugated rat immunoglobulin G1 and fluorescein isothiocyanate–conjugated rat immunoglobulin G1 (BD Biosciences, San Diego, CA) were used as isotype controls. After washing, the specimens were analyzed within 1 hour using an FACSCalibur flow cytometer (BD Biosciences).
The collected ADMSCs were then observed by microscope and analyzed by flow cytometry. About 5 × 105 ADMSCs were resuspended in 1 mL of PBS and incubated at 4°C for 30 minutes with the following antirat antibodies (all from BD Biosciences, San Jose, CA): phycoerythrin-conjugated CD29, CD44, and D-related human leukocyte antigen (HLA-DR); fluorescein isothiocyanate–conjugated CD31, CD45, and CD105. Phycoerythrin-conjugated rat immunoglobulin G1 and fluorescein isothiocyanate–conjugated rat immunoglobulin G1 (BD Biosciences, San Diego, CA) were used as isotype controls. After washing, the specimens were analyzed within 1 hour using an FACSCalibur flow cytometer (BD Biosciences).
8
Apoptosis Evaluation of Corpus Luteum
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For cell apoptosis evaluation, the corpus luteum was disassociated from rat ovaries on the specific day after pregnancy under dissecting microscope according to the method described by Care et al. (2013 (link)) with minor modifications. Briefly, the corpus luteum was trimmed of fat and minced into small pieces before enzymatic digestion into single cells. The minced tissues were incubated for 1 h at 37°C in DMEM F12 containing 0.1% collagenase A (Gibco), dispase (Gibco), and 25 μg/ml DNase 1 (Sigma-Aldrich). The cells were passed through a 70-μm nylon strainer (Becton Dickinson, Franklin Lakes, NJ, USA) to remove debris, and the filtrate was centrifuged at 300 g and 4°C for 5 min to pellet the suspended cells. After that, the cells were washed in FACS buffer (PBS containing 0.1% BSA). Thereafter, cells were re-suspended and stained by Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology, Haimen, China) according to the protocol provided by the manufacturer. The apoptotic cells were thereafter measured by using a BD FAC Symphony A5 system (Becton Dickinson, Franklin, NJ, USA).
9
Isolation and Culture of Rat Inguinal ADSCs
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ADSCs derived from the inguinal fat pad of 6-weeks SD rats. In brief, the capillaries were removed from the lipoaspirate, minced with phosphate-buffered solution (PBS, GenomSciences, Hangzhou, China) and digested with 1% collagenase A (Gibco, Carlsbad, United States) for 1 h at 37°C. After filtration, the mixture was centrifuged (1,000 rpm, 10 min) at room temperature (RT), and the supernatant was discarded. The collected cells were washed with PBS, centrifuged (1,000 rpm, 5 min) and then resuspended in DMEM/F12 medium (Gibco, Carlsbad, United States) with 10% fetal calf serum (FCS; Gibco, Carlsbad, United States) and 1% penicillin-streptomycin (PS) in a humidified 5% CO2 at 37°C; When the cells passed 3 generations, they were identified and used for next experiments.
10
Isolation of Rat Lung Endothelial Cells
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Rat lung endothelial cells were isolated from a dedicated cohort of rats as described previously. 59 In brief, animals were perfused with DMEM (cat. # 31053-028, Gibco-ThermoFisher Scientific, Waltham, MA, USA) and lungs were inflated with DMEM containing collagenase A at a final concentration of 2 mg mL -1 (cat. # 10103586001, Roche, Basel, Switzerland). Lung tissue was digested at 37°C with collagenase A (cat. # 10103586001, Roche, Basel, Switzerland) and DNAse (cat. # 18047-019, Invitrogen-ThermoFisher Scientific, Waltham, MA, USA); final concentrations of 2.5 mg μL -1 and 120 U mL -1 , respectively. Digested tissue material was then passed through a 40 μm cell strainer (cat. # 352340, BD Biosciences, San Jose, CA, USA) and centrifuged. Isolated cells were mixed with 30 μL of anti-CD31 precoated Dynabeads ® (cat. # 11155D, ThermoFisher Scientific, Waltham, MA, USA) and incubated on an orbital shaker for 20 minutes at 4°C. After washing, cells were lysed in RLT buffer (cat. # 74104, Qiagen, Hilden, Germany) and processed for RNA isolation and qPCR analysis.
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