Trap leukocyte kit

Manufactured by Merck Group

Sourced in United States

The TRAP-Leukocyte kit is a laboratory equipment product from Merck Group. It is designed to isolate and purify leukocytes from biological samples. The kit includes reagents and components necessary for the targeted extraction and enrichment of leukocytes.

Automatically generated - may contain errors

Lab products found in correlation

Osteo assay 96 well plates, by Corning (2 mentions) Model bx60f, by Olympus (1 mentions) Ti dh microscope, by Nikon (1 mentions) Fitc phalloidin, by Merck Group (1 mentions) Alpha mem, by Lonza (1 mentions) A1r confocal fluorescent microscope, by Nikon (1 mentions) Human m csf, by Thermo Fisher Scientific (1 mentions) α mem, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Equitech-Bio (1 mentions)

Spelling variants of this product

TRAP kit (138 citations) Leukocyte Acid Phosphatase (TRAP) kit (21 citations) Acid phosphatase leukocyte (TRAP) kit (387A) (6 citations) Leukocyte TRAP Kit 387-A (3 citations) Leukocytes acid phosphatase (TRAP) detection kit (2 citations)

8 protocols using trap leukocyte kit

Histological Analysis of Bone Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Femurs or tibias were fixed with 4% paraformaldehyde (PFA) overnight at 4°C and decalcified in 19% EDTA for 2 weeks. The tissues were then dehydrated through a graded ethanol series and embedded in paraffin. Longitudinal 5-µm paraffin sections were prepared for histological analysis and immunohistochemistry (IHC) or immunofluorescence (IF). Specifically, TRAP staining was carried out using acid phosphatase, Leukocyte TRAP Kit (Sigma-Aldrich, St. Louis, MO, USA). IHC was performed using antibodies mentioned above.
All histology and IHC/IF images were analyzed on a microscope (Model BX60F; Olympus Optical Co., Japan) equipped with a digital camera (Model 01-RET-OEM-F-CLR-12; QImaging, Surrey, Canada). Photomicrographs were taken with a Nikon Ti-DH Microscope. Images were processed in PhotoShop (Adobe, San Jose, CA, USA).

Zhao G., Huang B.L., Rigueur D., Wang W., Bhoot C., Charles K.R., Baek J., Mohan S., Jiang J, & Lyons K.M. (2018). CYR61/CCN1 Regulates Sclerostin Levels and Bone Maintenance. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 33(6), 1076-1089.

+ Open protocol

+ Expand

Osteoclast Differentiation and Bone Resorption

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

PBMCs were seeded at a density of 750,000 cells/cm² in alpha-MEM (Lonza) supplemented with 10% FCS, 2 mM L-glutamine and 1% P/S. Cells were left to adhere for 4 hours. Next, the medium was refreshed and supplemented with 25 ng/ml human M-CSF and 50 ng/ml human sRANKL (Peprotech). The culture medium was refreshed twice per week and cultures were stopped on day 14. RAW264.7-derived osteoclast cultures were established as described previously.^{19 (link)} TRAP activity in osteoclast cultures was detected using the Leukocyte TRAP kit (Sigma-Aldrich). Alternatively, cultures were lysed for RNA or protein extraction. Bone resorption by osteoclasts was assessed in Osteo Assay 96-well plates (Corning) as described previously.^{19 (link)} Actin ring formation was assessed by staining cultures with phalloidin-FITC (Sigma-Aldrich), followed by analysis on an A1R confocal fluorescent microscope (Nikon).

Muller J., Bolomsky A., Dubois S., Duray E., Stangelberger K., Plougonven E., Lejeune M., Léonard A., Marty C., Hempel U., Baron F., Beguin Y., Cohen-Solal M., Ludwig H., Heusschen R, & Caers J. (2018). Maternal embryonic leucine zipper kinase inhibitor OTSSP167 has preclinical activity in multiple myeloma bone disease. Haematologica, 103(8), 1359-1368.

+ Open protocol

+ Expand

Murine Osteoclast Differentiation and Quantification

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Murine primary (twelve-week old mice of both sexes) and RAW264.7-derived osteoclast cultures were established as described previously [41 (link)]. Of note, the osteoclast differentiation medium for both primary and cell line-derived cells was α-MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL P/S, 100 ng/mL MCSF//RANKL (primary) and 30 ng/mL RANKL (cell line). Osteoclasts were determined by tartrate-resistant acid phosphatase (TRAP) staining (Leukocyte TRAP kit; Sigma-Aldrich, St-Louis, MO, USA) following the supplier’s protocol. Bone resorption was assessed in Osteo Assay 96-well plates (Corning, Corning, NY, USA) as described previously [41 (link)] and quantified with ImageJ software (https://imagej.nih.gov/ij/).

Muller J., Duray E., Lejeune M., Dubois S., Plougonven E., Léonard A., Storti P., Giuliani N., Cohen-Solal M., Hempel U., Thijssen V.L., Beguin Y., Heusschen R, & Caers J. (2019). Loss of Stromal Galectin-1 Enhances Multiple Myeloma Development: Emphasis on a Role in Osteoclasts. Cancers, 11(2), 261.

+ Open protocol

+ Expand

Differentiation of Osteoclasts from Murine Bone Marrow

Check if the same lab product or an alternative is used in the 5 most similar protocols

The femur and tibia bones were extracted from adult mice and the bone marrow was flushed out with α‐MEM. The cells were dispersed and filtered through a 70 μm mesh, which were pelleted. The red blood cells were lyzed and the bone marrow cells were washed and cultured in α‐MEM containing 15% FBS, 100 μg/mL penicillin, 100 μg/mL streptomycin at 37°C for 16 hours. The nonadherent cells were harvested and seeded at 1 × 10⁶ cells/well in 96‐well plates for TRAP staining. These cells were cultured in α‐MEM medium containing M‐CSF (50 ng/mL) and RANKL (100 ng/mL) for 5 days, and then stained for TRAP using a TRAP‐leukocyte kit (Sigma‐Aldrich), following the manufacturer's instructions.

Li P., Deng Q., Liu J., Yan J., Wei Z., Zhang Z., Liu H, & Li B. (2018). Roles for HB‐EGF in Mesenchymal Stromal Cell Proliferation and Differentiation During Skeletal Growth. Journal of Bone and Mineral Research, 34(2), 295-309.

+ Open protocol

+ Expand

Osteoclast Differentiation from Bone Marrow Monocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Bone marrow cells were cultured in α-MEM supplemented with 100 units/mL penicillin/streptomycin and 10% FBS (v/) with 10 ng/mL M-CSF for 16 h to separate adherent cells from non-adherent cells. Non-adherent cells were harvested and used as enriched bone marrow–derived monocyte/macrophage precursors (i.e., BMMs). BMMs were further cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 4 days followed by fixation and TRAP staining using TRAP-Leukocyte kit (Sigma, St Louis, MO, USA). TRAP-positive cells containing more than three nuclei were considered multinucleated bona fide osteoclasts. In certain conditions, NM-cKO cells were either cultured with TNFα neutralizing antibody (0.2 μg/mL) or transfected with retroviral pMX-WT-NEMO generated by transfecting pMX-WT-NEMO in Plat-E cells as described previously28 (link).

Swarnkar G., Shim K., Nasir A.M., Seehra K., Chen H.P., Mbalaviele G, & Abu-Amer Y. (2016). Myeloid Deletion of Nemo Causes Osteopetrosis in Mice Owing to Upregulation of Transcriptional Repressors. Scientific Reports, 6, 29896.

+ Open protocol

+ Expand

Quantifying Multinucleated Osteoclasts

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were washed with PBS and fixed with 10% formalin. TRAP⁺ cells were stained using a TRAP leukocyte kit (Sigma-Aldrich). The staining was performed according to manufacturer’s instructions, except the following adaptation: to visualize osteoclasts specifically, we used 1 M tartrate solution instead of the 0.3 M recommended by the manufacturer. Per well, 7 photos were taken at different locations to minimize the effect of unequal osteoclast development across the wells. Osteoclasts with ≥ 3 nuclei were counted using the online available ImageJ software (https://imagej.net/Welcome).

Razawy W., Alves C.H., Koedam M., Asmawidjaja P.S., Mus A.M., Oukka M., Leenen P.J., Visser J.A., van der Eerden B.C, & Lubberts E. (2021). IL-23 receptor deficiency results in lower bone mass via indirect regulation of bone formation. Scientific Reports, 11, 10244.

+ Open protocol

+ Expand

Osteoclastogenesis Assay and Gene Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

For Osteoclastogenesis assay, 50 thousands whole bone marrow cells (WBMs) were counted and cultured in 96-well tissue culture plate with α-MEM (Invitrogen, Grand Island, NY, USA) containing 10% heat-inactivated FBS (Equitech-Bio, Kerrville, TX, USA), in the presence of macrophage colony-stimulating factor (M-CSF) (20 ng/mL) and receptor activator of nuclear factor kappa-B ligand (RANKL) (50 ng/mL). After 5 days, mature osteoclasts were fixed and stained for TRAP-positive Multinuclear cells by using TRAP-Leukocyte kit (Sigma, St. Louis, MO). For assay of osteoclastogenesis specific genes, 200 thousands of whole bone marrow free of red blood cells were cultured in osteoclastogenesis media for 3 days and cells were lysed for mRNA isolation. In case of osteoclastogenesis from IECs colcutured with WBMs, 20K of sorted CD326+IECs infected with lentivirus containing IKK2ca construct or control overnight (for 16h). Infected IECs were cocultured with 50K of wild-type (WT)/WBMs in the absence or presence of permissive levels of RANKL (1ng/ml) and M-CSF. After 5 days, cells were fixed and proceed for TRAP staining to identify TRAP+MNCs formation. Additionally, infected IECs were also designed to cocultured with sorted CD3e-CD19-CD45+ILC population for 48 h to investigate ILC population by flow cytometry.

Ke K., Chen T.(., Arra M., Mbalaviele G., Swarnkar G, & Abu-Amer Y. (2019). Attenuation of NF-κB in intestinal epithelial cells is sufficient to mitigate the bone loss co-morbidity of experimental mouse colitis.. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 34(10), 1880-1893.

+ Open protocol

+ Expand

Isolation and Differentiation of Bone Marrow-Derived Macrophages

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total bone marrow cells were isolated from the long bones (femur and tibia) and cultured in α-MEM media supplemented with 100 units/mL penicillin/streptomycin and 10% FBS (v/v) with 10 ng/mL M-CSF overnight to separate the adherent cells. One day after isolation, the non-adherent cells were collected and used as enriched bone marrow–derived macrophage (BMMs). BMMs were further cultured with M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 4 days followed by fixation and TRAP staining using TRAP-Leukocyte kit (Sigma, St Louis, MO, USA). To investigate changes in autophagy, BMMs were cultured in M-CSF (20 ng/mL) and RANKL (50 ng/mL) for 2 days and used as pre-osteoclast (preOC) for different experiments. To investigate the effect of exogenous expression of different NEMO, NEMO mutants and ISG15, the BMMs after one day of isolation, were transduced with retroviral particles (generated using PLAT-E cells) and osteoclast differentiation was initiated after 2 day of viral transduction.

Adapala N.S., Swarnkar G., Arra M., Shen J., Mbalaviele G., Ke K, & Abu-Amer Y. (2020). Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO. eLife, 9, e56095.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!