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10 protocols using clspa

1

Isolation of Adipocytes from Bone Marrow and Subcutaneous Fat

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Bone marrow biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin; Invitrogen) and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml in complete RPMI medium) for 1 h at 37 °C. After centrifugation for 10 min at 150 g with low break and before ficoll purification, adipocytes formed a floating layer on the top of the medium which was carefully transferred into a fresh 15ml tube. BM biopsies were then centrifuged and BMMCs purified by density gradient centrifugation (Ficoll-Hypaque). Subcutaneous fat tissue was placed into a petri dish and reduced into small fragments of approximately 1 mm with a scissor. Small fragments were transferred into a 15 ml Falcon and filled with PBS + 2% FCS to 14 ml. For digestion 200 μl of collagenase (Worthington CLSPA #L5005273; Stock 1000 U/ml; Final concentration 200 U Collagenase) was added and incubated for 1 h at 37 °C. The digested suspension was passed through a 100 μM cell strainer and afterwards collected in a 15 ml tube. BM and subcutaneous adipocytes were treated the same. Both 15 ml tubes containing adipocytes were centrifuged at 150 g for 10 min and afterwards, adipocytes were isolated by flotation. Informed consent for test and publication was given and documented from each patient.
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2

Isolation of Primary Salivary Gland Epithelial Cells

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Primary salivary gland epithelial cells were isolated from adult
male Sprague–Dawley rat submaxillary glands. Glands were removed
aseptically; the outer connective tissue was excised followed by washing
in Hank’s balanced salt solution (HBSS) supplemented with 1×
antibiotic/antimycotic (Life Technologies 100× antibiotic–antimycotic
solution, Grand Island, NY). Glands were cut into cubes of approximately
1 mm3 followed by washing 2× in HBSS using gravity
settling. Cubes were then suspended in HBSS supplemented with collagenase
(CLSPA, 150 units/mL) and hyaluronidase (150 units/mL) (Worthington
Biochemical Corporation, Lakewood, NJ) and dissociated for 45 min
at 37 °C with gentle rocking. The digests were passed through
a 70 μm nylon mesh, and isolated cells were collected by centrifugation
at 1300 rpm for 5 min. Cell pellets were resuspended in advanced DMEM:F12
supplemented with 2.5% FBS, 2 mM Glutamax, 100 units/mL penicillin,
100 units/mL streptomycin, 0.25 μg/mL fungizone, and 10 ng/mL
epidermal growth factor (EGF). Cells were subcultured by trypsinization
and used at passage 3.
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3

Isolation of Peripheral and Bone Marrow Mononuclear Cells

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Peripheral blood mononuclear cells from heparinized blood were purified by Ficoll-Hypaque density gradient centrifugation (GE Healthcare Life Sciences). Cells were washed with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin; Invitrogen). PBMCs from cohort A were frozen according to a standard protocol using 10% DMSO as cryoprotective agent. PBMCs from Cohort B and C were freshly used.
Bone marrow samples (cohort C) were obtained from the femur shaft of patients of varying age (Table 1) during hip replacement surgery. A biopsy of Substantia spongiosa ossium, which would otherwise have been discarded, was used to isolate BM mononuclear cells (BMMCs). BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin; Invitrogen), and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs purified by density gradient centrifugation (Ficoll-Hypaque). BMMCs were freshly used.
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4

Isolation of PBMCs and ATMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Adipose tissue mononuclear cells (ATMCs) were isolated from intra-abdominal fat samples, which were first dissected then dissociated into a single cell suspension using a GentleMACS device (Miltenyi). Samples were digested in 2 ml RPMI medium (Gibco) supplemented with 20 U/ml CLSPA (Worthington) for every 4 g fat, over 1 h at 37 °C. The resulting cell suspension was washed in RPMI, and the fat layer that formed after centrifugation was decanted before the cell pellet was resuspended and filtered through a 40-µm nylon mesh. The cell suspension was then separated by Ficoll density gradient centrifugation. Isolated PBMCs and ATMCs were stored in cryopreservation medium at 106 cells/100 µl [70% FCS (Gibco), 20% RPMI, 10% DMSO] in liquid nitrogen.
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5

Isolation of BMMCs from Healthy Individuals

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Human BM and blood samples were obtained from systemically healthy individuals who did not receive immunomodulatory drugs or suffer from diseases known to influence the immune system, including autoimmune diseases and cancer. Hip replacement surgery was performed and bone from the femur shaft was harvested. A biopsy of substantia spongiosa osseum, which would otherwise have been discarded, was used to isolate BMMCs. BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin; Invitrogen) and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/mL in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs purified by density gradient centrifugation (Ficoll‐Hypaque). Experiments were performed using BMMCs and not with all BM cells in order to obtain cleaner populations and thus to improve the quality of the results. qPCR experiments comparing the expression of effector/memory cell survival factors in BMMCs and in BM cells showed similar results (data not shown). PBMCs were used for pilot and stimulation control experiments. Purification of PBMCs from heparinized blood was also performed by density gradient centrifugation.
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6

Isolation of Bone Marrow Mononuclear Cells

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Paired BM and blood samples were obtained from 55 systemically healthy individuals (20 females, 25 males, age range 36–89 years, mean age 64 ± 12.1 years) who did not receive immunomodulatory drugs or suffer from diseases known to influence the immune system, including autoimmune diseases and cancer. Bone from the femur shaft was harvested during hip replacement surgery, and a biopsy of substantia spongiosa osseum, which would otherwise have been discarded, was used to isolate BMMCs. BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin; Invitrogen), and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/mL in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs were purified by density gradient centrifugation. Fresh BMMCs and PBMCs were used for the experiments.
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7

Actin cytoskeleton dynamics in egg chambers

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Egg chambers were dissected in live imaging medium, and then incubated with collagenase (1,000 Units ml−1 CLSPA; Worthington Biochemical Corp.) dissolved in the final volume of 100 μl culturing medium for 20 min before being mounted for imaging. For the chemical inhibition of Rho1 activity, dissected egg chambers were incubated with two Rho inhibitors: C3 exoenzyme61 (link) (Enzo Life Sciences) at 10 μg μl−1 or Rhosin62 (link) (Merk) at 250 μM for 20 min before being mounted for imaging.
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8

Isolation of Bone Marrow and Peripheral Blood Mononuclear Cells

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Samples were obtained from systemically healthy individuals who do not suffer from diseases known to affect the immune system. All samples were obtained from people undergoing elective surgeries because of osteoarthrosis. The donors comprised of 95 individuals aged between 39 and 87 (mean age: 67.45 ± 10.95, mean BMI: 27.9 ± 5.03, sex: 50 F, 46 M). The number of samples used in individual experiments are given in the figures and legends.
For the isolation of bone marrow mononuclear cells (BMMCs), a fragment of substantia spongiosa osseum, which would otherwise be discarded was collected during routine hip replacement surgery. The bone was further fragmented and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml) in complete RPMI medium (RPMI 1640; Corning supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin; Sigma) for 1 h at 37 °C. BMMCs were extracted using a filtered tube centrifugation step, and then purified using density gradient centrifugation (Lymphoprep®; Stemcell technologies). Heparinised blood from the same donors was collected, and peripheral blood mononuclear cells (PBMCs) were also purified by density gradient centrifugation.
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9

Isolation of BM and PB Mononuclear Cells

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Hip replacement surgery was performed and bone from the femur shaft was harvested. A biopsy of substantia spongiosa osseum, which would otherwise have been discarded, was used to isolate BM mononuclear cells (BMMCs). BM biopsies were fragmented, washed once with complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin; Invitrogen) and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml in complete RPMI medium) for 1 h at 37°C. BM biopsies were then centrifuged and BMMCs purified by density gradient centrifugation (Ficoll-Hypaque). Purification of PB mononuclear cells (PBMCs) from heparinized blood was also performed by density gradient centrifugation.
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10

Isolation of Bone Marrow and Peripheral Blood Mononuclear Cells

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Samples were obtained from systemically healthy individuals who did not suffer from diseases known to affect the immune system. The donors comprised of 72 individuals (37 females, 35 males) aged between 31 and 89 years (mean age: 69.7 ± 12.9) and BMI between 20.2 and 43.5 (mean BMI: 28.9 ± 5.4) The number of samples used in individual experiments are given in the figures and legends. For the isolation of bone marrow mononuclear cells (BMMCs), a fragment of substantia spongiosa osseum, which would otherwise be discarded, was collected during routine hip replacement surgery. The bone was further fragmented and treated with purified collagenase (CLSPA, Worthington Biochemical; 20 U/ml) in complete RPMI medium (RPMI 1640, Corning supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin, Sigma) for 1 h at 37 °C. BMMCs were extracted using a filtered tube centrifugation step, and then purified using density gradient centrifugation (Lymphoprep®, Stemcell technologies). Paired samples of heparinised blood from the same donors were collected, and peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation.
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