Calvaria cells were isolated from 3‐ to 4‐day‐old C57BL/6 mice as described.(35 ) To obtain bone marrow–derived stromal cells, total bone marrow cells pooled from 3–4 C57BL/6 female mice at 2–4 months of age were cultured with 20% FBS, 1% penicillin–streptomycin–glutamine, and 50 μg/ml of ascorbic acid (Sigma) in 10‐cm culture dishes for 5 days. Half of the medium was replaced every 3 days. Cells were then cultured with 10% FBS, 1% penicillin–streptomycin–glutamine, 50 μg/ml of ascorbic acid, and 10mM β‐glycerophosphate (Sigma0Aldrich) for 21 days with or without HBX. Mineralized matrix was stained with 40mM Alizarin Red solution (Sigma‐Aldrich). BrdU incorporation was measured with a Cell Proliferation ELISA Chemiluminescence Kit (Roche Diagnostics). Alkaline phosphatase activity was determined in cells cultured for 7 days, and cells were lysed in 100mM glycine, 1mM MgCl2, and 1% Triton X‐100 at pH 10 using a buffer containing 2‐amino‐2‐methylpropanol and p‐nitrophenylphosphate (Sigma‐Aldrich). Alkaline phosphatase activity was normalized to total protein concentration, which was measured using a detergent‐compatible kit (Bio‐Rad). For all assays, cells were plated in triplicate.
Detergent compatible kit
Manufactured by Bio-Rad
Sourced in United States
The Bio-Rad detergent-compatible kit is a laboratory product designed to facilitate protein quantification in the presence of detergents. It provides a reliable method for determining protein concentrations in samples containing interfering substances, such as detergents, which can otherwise disrupt traditional protein assays.
Automatically generated - may contain errors
Lab products found in correlation
P nitrophenyl phosphate, by Merck Group (3 mentions)
2 amino 2 methylpropanol, by Merck Group (3 mentions)
Fl 500, by Agilent Technologies (2 mentions)
E06 igm, by Avanti Polar Lipids (2 mentions)
Alizarin red solution, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Caspase glo 9 assay, by Promega (1 mentions)
Oxldl, by Thermo Fisher Scientific (1 mentions)
11 protocols using detergent compatible kit
1
Isolation and Characterization of Murine Bone Cells
2
Retinal Protein Extraction and Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized with carbon dioxide asphyxiation and PBS-perfused. Retinas were dissected and snap-frozen in liquid nitrogen. Both retinas from each animal were homogenized in 200 μL Holt’s lysis buffer and sonicated, then spun at 500 g for 5 min at 4 °C, and the supernatant was collected. Protein was quantified using a BioRad detergent compatible kit (Hercules, CA) according to the manufacturer’s protocol. The volume equivalent to 60 μg protein was taken for analysis, and 8 pmol of bovine serum albumin were added as a non-endogenous internal standard. Samples were mixed, heated and cooled to room temperature. Acetone (1 mL) was added and proteins were precipitated overnight at –20 °C. The resultant pellet was reconstituted in 60 μL of sample loading buffer and a 20 μL aliquot was resolved by short-run gel electrophoresis (1.5 cm into gel), gel-fixed, and stained. Each lane was cut as a single sample, washed, reduced with DTT, alkylated with idoacetamide, and digested overnight with trypsin. The peptides from the digestion were extracted, evaporated to dryness, and reconstituted in 150 μL 1% acetic acid for analysis.
3
Retinal Protein Extraction and Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice (n = 6 mice/group, 28 weeks diabetic) were euthanized with carbon dioxide asphyxiation and PBS-perfused. Retinas were dissected and snap-frozen in liquid nitrogen. Both retinas from each animal were pooled, homogenized in 200 μL Holt’s lysis buffer and sonicated and then spun 500 xg for 5 min at 4°C, and supernatant was collected. Protein was quantified using a BioRad detergent compatible kit (Hercules, CA) according to manufacturer’s protocol. The volume equivalent to 60 μg protein was taken for analysis, and 8 pmol bovine serum albumin added as a non-endogenous internal standard. Samples were mixed, heated and cooled to room temperature. 1 mL of acetone was added and proteins precipitated overnight at -20°C. The resultant pellet was reconstituted in 6 0μL of sample loading buffer and a 20 μL aliquot resolved by short-run gel electrophoresis (1.5 cm into gel), and gel fixed and stained. Each lane was cut as a single sample, washed, reduced with DTT, alkylated with idoacetamide and digested overnight with trypsin. The peptides from the digestion were extracted, evaporated to dryness and reconstituted in 150 μL 1% acetic acid for analysis.
4
Caspase-9 and Caspase-3 Activity Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caspase-9 activity was measured by the Caspase-Glo® 9 Assay (G8210, Promega), according to the manufacturer’s instructions. Briefly, BMMs were cultured in a 96-well white-wall tissue culture plate with RANKL and E2 for 24h. Cell culture media was replaced with 100 µl of Caspase-Glo® 9 reagent. Contents were mixed for 30 seconds and incubated for 30 min at room temperature. The luminescence signal was measured in a Cytation™ 5 microplate reader.
Caspase-3 activity was determined by measuring the degradation of the fluorescent substrate DEVD-AFC (Biomol Research Labs, Plymouth, PA) and protein concentration was measured by a Bio-Rad detergent–compatible kit (Bio-Rad, Hercules, CA), as described previously (10 (link)). Briefly, cultured BMMs were lysed with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 5 mg ml-1 leupeptin, 0.14 U ml-1 aprotinin, 1 mM phenylmethylsulfonylfluoride, and 1% Triton X-100. Cell lysates were then transferred to a new plate and incubated with 50 mM DEVD-AFC in 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, and 10% glycerol. The released fluorescent signal (λex/em =400/510 nm) was measured kinetically in a Cytation™ 5 microplate reader.
Caspase-3 activity was determined by measuring the degradation of the fluorescent substrate DEVD-AFC (Biomol Research Labs, Plymouth, PA) and protein concentration was measured by a Bio-Rad detergent–compatible kit (Bio-Rad, Hercules, CA), as described previously (10 (link)). Briefly, cultured BMMs were lysed with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 5 mg ml-1 leupeptin, 0.14 U ml-1 aprotinin, 1 mM phenylmethylsulfonylfluoride, and 1% Triton X-100. Cell lysates were then transferred to a new plate and incubated with 50 mM DEVD-AFC in 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, and 10% glycerol. The released fluorescent signal (λex/em =400/510 nm) was measured kinetically in a Cytation™ 5 microplate reader.
5
Alkaline Phosphatase Activity Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the alkaline phosphatase (ALP) activity measurement, the cells were lysed in 100mM glycine, 1mM MgCl2, and 1% Triton X‐100 at pH 10 using a buffer containing 2‐amino‐2‐methylpropanol and p‐nitrophenylphosphate (Sigma‐Aldrich). ALP activity was normalized to total protein concentration measured using Bio‐Rad detergent‐compatible kit (Bio‐Rad Laboratories). For all assays, triplicate cultures were analyzed.
6
Quantitative Caspase-3 Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We lysed the cells in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 5 mg ml−1 leupeptin, 0.14 U ml−1 aprotinin, 1 mM phenylmethylsulfonylfluoride and 1% Triton X-100. Lysates (100 mg protein) were incubated with 50 mM DEVD-AFC in 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA and 10% glycerol. Caspase-3 activity was measured by determining the degradation of the fluorometric substrate DEVD-AFC. We measured the released fluorescent in a microplate fluorescence reader with excitation/emission wavelengths of 340/542 nm. We measured protein concentration using a Bio-Rad detergent-compatible kit (Bio-Rad).
7
Quantifying Osteoclast Apoptosis in Response to WNT3A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteoclasts derived from purified non-adherent bone marrow osteoclast precursors isolated from 3 animals for each genotype were treated with WNT3A once osteoclasts had developed in the culture plate in triplicates. After 24 hours of treatment, the cultures were fixed and subject to TUNEL and TRAP staining. The total number of osteoclasts and the number of apoptotic osteoclasts in the plate were quantified. Osteoclasts were considered apoptotic when at least one of their nuclei was TUNEL positive. The TUNEL method was performed using the FragEL DNA fragmentation detection kit (EMD Chemicals, San Diego, CA) before staining for TRAP. Multinuclear TRAP-positive and TUNEL-positive cells were enumerated. Caspase-3 activity was measured by determining the degradation of the fluorometric substrate DEVD (Biomol Research Laboratories, Plymouth Meeting, PA), and protein concentration was measured using a Bio-Rad detergent-compatible kit (Bio-Rad Laboratories, Hercules, CA).
8
Quantifying Apoptosis via Caspase-3 Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptotic cells were quantified by measuring caspase 3 activity in cell lysates as described.(16 ) Caspase 3 activity was measured by determining the degradation of the fluorometric substrate DEVD‐AFC, which contains the amino acid sequence of the caspase 3 cleavage site DEVD (Asp‐Glu‐Val‐Asp), conjugated with 7‐amino‐4‐trifluoromethylcoumarin (AFC). Briefly, lysates (100 μg protein) were incubated with 50μM DEVD‐AFC in 50mM zwitterionic sulfonic acid (HEPES) (pH 7.4), 100mM NaCl, 0.1% 3‐ ((3‐Cholamidopropyl)dimethylammonio)‐1‐Propanesulfonic Acid (CHAPS), 10mM dithiothreitol (DTT), 1mM EDTA, and 10% glycerol. The released fluorescent AFC was measured in a microplate fluorescence reader FL500 (BioTek Instruments, Winooski, VT, USA) with excitation/emission wavelengths of 400/510 nm. Protein concentration in the lysate was measured using a Bio‐Rad detergent–compatible kit (Bio‐Rad Laboratories, Hercules, CA, USA). OxLDL was obtained from Alfa Aesar (Alpha Aesar, Haverhill, MA, USA) and E06 IgM from AVANTI Polar Lipids Inc. (Alabaster, AL, USA; Cat Number 330001‐100 μg, lot number E06‐17 and E06‐19).
9
Caspase-3 Activity Assay in BMMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caspase-3 activity was measured by determining the degradation of the fluorometric substrate DEVD-AFC (Biomol Research Labs, Plymouth, PA) and protein concentration was measured using a Bio-Rad detergent–compatible kit (Bio-Rad, Hercules, CA), as described previously33 (link),41 (link). In brief, BMMs were plated on 96-black well plates with M-CSF (30 ng/ml) and RANKL (30 ng/ml) in α-MEM complete media with or without E2 (10−8 M). After 24 h, the cultured cells were lysed the cells in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 5 mg ml−1 leupeptin, 0.14 U ml−1 aprotinin, 1 mM phenylmethylsulfonylfluoride, and 1% Triton X-100. Cell lysates were incubated with 50 mM DEVD-AFC in 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, and 10% glycerol. The released fluorescent signal was measured in a microplate fluorescence reader with excitation/emission wavelengths of 340/542 nm.
10
Caspase 3 Assay for Apoptosis Quantification
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!