To isolate pancreatic acinar cells, mice were euthanized and pancreas were collected and immediately washed with ice-cold HBSS twice. Pancreas was transferred to an eppendorf, well-minced, and then transferred into a 50 ml tube. Centrifuged for 2 min at 450 × g. The supernatant was discarded to remove cell debris and blood cells. 5 ml of collagenase IA solution (HBSS containing 10 mM HEPES, 200 U/ml collagenase IA (Sigma #C2674), and 0.25 mg/ml of trypsin inhibitor (Sigma #T6522) was added and incubated for 20 min at 37 °C with 200 rpm shaking. The enzymatic reaction was stopped by adding 10 ml of cold HBSS with 5% FBS and then centrifuged for 2 min at 450 × g, 4 °C. The supernatant was aspirated to remove collagenase IA solution. Cell pellets were resuspended, washed twice with 10 ml of cold HBSS with 5% FBS and filtered through a 100 μm strainer (Falcon) to remove non-digested materials. The cells were suspended in Waymouth’s medium and transferred to 6-well plate and placed for 24 h in a 37 °C humidified 5% CO2 incubator. Purity of acinar cells was examined by PNA staining (Vector Laboratories, #FL-1071) via flow cytometry as described. To avoid acinar cells undergoing acinar-to-ductal metaplasia, fresh acinar cells were used in most of the experiments. If acinar cells were to be treated for 16 h, level of ductal cell marker CK19 was measured.
Collagenase ia
Manufactured by Merck Group
Sourced in United States, France, United Kingdom, Germany, Japan
Collagenase IA is a highly purified enzyme used for the dissociation and isolation of cells from a variety of tissues, including adipose, cardiac, and skin tissues. It is effective in breaking down the collagen present in the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Trypsin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dnase 1, by Roche (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dynabead, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Worthington (4 mentions)
Amphotericin b, by Thermo Fisher Scientific (3 mentions)
Percoll, by Cytiva (3 mentions)
Hyaluronidase, by Merck Group (3 mentions)
Dmem f12, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Pronase, by Merck Group (2 mentions)
80 protocols using collagenase ia
1
Isolating Pancreatic Acinar Cells from Mice
2
Isolation of Ovine Adipose-Derived MSCs
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Adipose tissue-derived mesenchymal stem cells were isolated from the adipose tissue of 3-month-old female lambs taken at the slaughterhouse. Adipose tissue was sterilely removed from the animal and placed in sterile PBS (Phosphate Buffed Saline, Gibco, UK), supplemented with antibiotics (100 IU Penicillin, 100 μg/mL Streptomycin, and 0.25 μg/mL Amphotericin B—all purchased from Gibco, UK), and quickly transported to the laboratory. The tissue was minced into small fragments and subsequently digested with Collagenase IA (Sigma-Aldrich, Gillingham, UK) at 0.1% in PBS (Phosphate Buffed Saline, Gibco, UK) at 37 °C for 60 min with slow stirring. At the end of the digestion, DMEM (Gibco, UK) was added with 10% FCS (Fetal calf serum, Gibco, UK) and subsequently centrifuged at 1200 g for 10 min. The pellet was washed 3 times in PBS, re-suspended in DMEM with 10% FCS, seeded in 75 cm2 flasks, and incubated at 37 °C with 5% CO2. DMEM with 10% FCS was replaced every 2 days, and cells were observed daily with an inverted light microscope (Nikon eclipse TE2000-U).
3
Kidney Cell Isolation Protocol
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Kidneys were perfused, minced, and placed in DMEM (Life Technologies, Carlsbad, CA, USA) containing 1 mg/ml collagenase IA and 100 ng/ml RNAse I (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min. Digested kidney tissues were passed through a 40-μm cell strainer (BD Falcon, San Diego, CA, USA), and the cell suspension obtained was centrifuged at 300 × g for 10 min. Cells were washed in PBS containing 2% BSA, suspended in 36% Percoll (Amersham Pharmacia Biotech, Little Chalfont, UK), and gently overlaid onto 72% Percoll. After centrifugation at 900 × g for 30 min at RT, cells were retrieved from the Percoll interface and washed twice in DMEM and once with staining buffer (PBS containing 2% BSA and 0.1% sodium azide).
4
Isolation and Expansion of hADSC
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hADSC were obtained as previously described (Zuk et al., 2001 (link); Baptista et al., 2009 (link)). Briefly, fragments and lipoaspirates were enzymatically digested with 10 mg/ml collagenase IA (Sigma) for 1 h at 37°C under agitation. Cells were plated at 1−2×104 cells/cm2 in DMEM (LGC) supplemented with 10% fetal bovine serum (Cultilab) and penicillin/streptomycin (Sigma), and kept overnight at 37°C, 5% CO2. Non-adherent cells were removed, and adherent cells were maintained as above and expanded in up to 6 passages.
After in vitro expansion, the hADSC used here were homogeneous for the expression of surface antigens described in the literature (including CD105, CD90, CD13 and CD44) and were negative for hematopoietic antigens (including CD45, CD14, CD34, CD3 and CD19), as previously reported by one of the authors (Baptista et al., 2007 ).
After in vitro expansion, the hADSC used here were homogeneous for the expression of surface antigens described in the literature (including CD105, CD90, CD13 and CD44) and were negative for hematopoietic antigens (including CD45, CD14, CD34, CD3 and CD19), as previously reported by one of the authors (Baptista et al., 2007 ).
5
Chondrocyte Isolation from Knee Articular Cartilage
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Human articular cartilage from the knee of a patient undergoing total knee
arthroplasty was processed for chondrocyte isolation. Briefly, the cartilage
tissue was aseptically dissected from the joint, minced, and washed with
Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies). Then, cartilage
was incubated for 30 min with a 0.5 mg/ml hyaluronidase (Sigma-Aldrich) solution
and for 1 h with a 1 mg/ml pronase (Merck) solution in a shaking water bath at
37°C. After that, cartilage fragments were washed with DMEM and incubated with a
0.5 mg/ml collagenase-IA (Sigma-Aldrich) solution in a shaking water bath at
37°C overnight. The resulting cell suspension was filtered with a 70-µm cell
strainer (BD Biosciences) to remove any undigested tissue, and collagenase was
rinsed off with DMEM containing 10% foetal bovine serum (FBS; Invitrogen SA).
Finally, the cell suspension obtained was transferred in DMEM supplemented with
10% FBS and 50 µg/ml ascorbic acid (Sigma-Aldrich) to a 75-cm2 tissue
culture flask (Nunc) and maintained at 37°C, in a humidified atmosphere under 5%
CO2. The culture medium was replaced every 2 days, and cells were
allowed to grow until subconfluence. Then, cells were harvested by
trypsinisation and counted with a haemacytometer for experiments on PLLA.
arthroplasty was processed for chondrocyte isolation. Briefly, the cartilage
tissue was aseptically dissected from the joint, minced, and washed with
Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies). Then, cartilage
was incubated for 30 min with a 0.5 mg/ml hyaluronidase (Sigma-Aldrich) solution
and for 1 h with a 1 mg/ml pronase (Merck) solution in a shaking water bath at
37°C. After that, cartilage fragments were washed with DMEM and incubated with a
0.5 mg/ml collagenase-IA (Sigma-Aldrich) solution in a shaking water bath at
37°C overnight. The resulting cell suspension was filtered with a 70-µm cell
strainer (BD Biosciences) to remove any undigested tissue, and collagenase was
rinsed off with DMEM containing 10% foetal bovine serum (FBS; Invitrogen SA).
Finally, the cell suspension obtained was transferred in DMEM supplemented with
10% FBS and 50 µg/ml ascorbic acid (Sigma-Aldrich) to a 75-cm2 tissue
culture flask (Nunc) and maintained at 37°C, in a humidified atmosphere under 5%
CO2. The culture medium was replaced every 2 days, and cells were
allowed to grow until subconfluence. Then, cells were harvested by
trypsinisation and counted with a haemacytometer for experiments on PLLA.
6
Isolation of Porcine Interstitial Macrophages
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The protocol of IM isolation was adapted from rat pulmonary interstitial macrophages preparation (Cong et al., 2002 (link)). To improve the purity of IM cells, pigs were euthanized by exsanguination and pulmonary vasculature was perfusated. Finally, the alveolar macrophages were collected from lungs followed by IM cells isolation. The lung tissue was chopped into pieces of less than 1 mm3 using scissors. To remove the remaining PAM cells and blood cells, the tissue was washed with PBS over a 100 μm cell strainer until the filtrate appeared to be clear. The tissue was then digested using 0.025% collagenase IA (Sigma-Aldrich, St Louis, MO, USA) for 60 min at 37°C in a shaking water bath. The digestion was filtrated by 200 mesh stainless steel filter, centrifuged at 1500 rpm for 10 min and re-suspended in RPMI 1640 culture medium with 10% FBS in Petri dishes. Following adherence to Petri dishes, the adherent cells were collected, washed, counted and recorded for viability and plated as PAM cells.
7
Isolation and Culture of Human Endometrial Stromal Cells
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HESCs were obtained at the time of hysterectomy for uterine fibroids from normally cycling premenopausal women. Patients were not undergoing hormonal treatment at the time of surgery. All the samples were collected during the proliferative phase of the cycle. HESCs were isolated and cultured as previously described30 (link)31 (link)32 (link). Briefly, endometrial samples were collected in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 containing Antibiotic-Antimycotic solution (Invitrogen). After enzymatic digestion by DNase I (Sigma) and Collagenase I a (Sigma), the stromal cells were separated from epithelial cells and passed into culture. Proliferating HESCs were cultured in maintenance medium of DMEM/F-12 containing 10% dextran-coated charcoal-treated FBS and 1% antibiotic–antimycotic solution (Life technologies). Confluent monolayers of HESCs were treated with or without 0.5 mM 8-bromo-cyclic adenosine monophosphate (8-br-cAMP; Sigma) and 10−6 M medroxyprogesterone acetate (MPA; Sigma). All experiments were conducted before the third passage of the cultures.
8
Isolation and Culture of Schwann Cells
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The isolation of SC was conducted according to previously reported method [27 (link)]. Typically, the STZ-induced diabetic model rats were anaesthetized using intraperitoneal injection of 4% pentobarbital sodium (1 mL/kg body weight). The bilateral sciatic nerves were isolated under sterile condition. After removing epineurium, the sciatic nerves were then cut with scissors into pieces. To attain dispersed cells, the small nerve fragments were digested with 1% collagenase IA (Sigma) and 0.5% trypsin (Sigma) in serum-free αMEM for 1 h at 37°C. Then, the DMEM/F-12 (Gibco) media containing 10% fetal bovine serum, 50 U/mL penicillin, 40 mg/mL streptomycin, and 0.3 mg/mL L-glutamine were added in order to cease digestion. The obtained mixtures were then filtrated through an 80 mesh sieve and cells were collected by centrifugation. Upon being resuspended by fresh culture media, the cells were seeds at 1.5 × 105 cells/well into 24-well plates precoated with poly-L-lysine (0.1 mg/mL, Sigma). The high glucose condition was made by supplementing extra glucose (30 mM). The cells, which exhibited anti-S-100 immunofluorescence, were identified as SC.
9
Isolation and Culture of Rat Nodose Ganglion Neurons
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The nodose ganglia were excised from anesthetized rats and placed in ice-cold Ca2+- and Mg2+-free Hank’s balanced salt solution (Gibco, Grand Island, NY, USA) supplemented with penicillin and streptomycin (100 U/mL and 100 μg/mL, respectively; Gibco). The ganglia were cleaned of connective tissue and desheathed, and then were enzymatically dissociated by incubation in HEPES-buffered DMEM/F-12 (DMEM/F-12; Gibco) containing collagenase Ia (1 mg/mL; Sigma-Aldrich), dispase II (1 mg/mL; Sigma-Aldrich), and DNase I (100 U/mL; Sigma-Aldrich) at 37°C for 40 minutes. Thereafter, they were mechanically dissociated by passing 5–10 times through a fire-polished glass Pasteur pipette before separation with a Percoll PLUS (Sigma-Aldrich) density gradient. Individual cells were harvested by centrifugation at 700 × g for 3 minutes. They were then plated onto coverslips precoated with poly-L-lysine (Sigma-Aldrich) and were incubated overnight at 37°C in DMEM/F-12 with 10% fetal bovine serum (Gibco) and antibiotics in a humidified 5% CO2 atmosphere.
10
Murine Pancreatic Acini Isolation
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Briefly, 5-week-old mice pancreata were immediately removed after euthanasia, cut into small sections, and digested with collagenase IA (Sigma-Aldrich) for 10 min at 37 °C as previously described49 (link). Acini were collected by centrifugation at 30 g for 5 min, washed and resuspended in Seahorse medium, and plated in pre-coated Cell Tak (Corning) Seahorse plates (Agilent).
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