Acid phosphatase leukocyte trap kit 387a

Manufactured by Merck Group

Sourced in United States

The Acid phosphatase leukocyte (TRAP) kit (387A) is a laboratory reagent designed for the detection and quantification of tartrate-resistant acid phosphatase (TRAP) activity in biological samples. TRAP is an enzyme found in certain types of cells, including leukocytes. The kit provides the necessary components to measure TRAP levels using a colorimetric assay.

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Lab products found in correlation

Rpmi 1640, by Thermo Fisher Scientific (3 mentions) Mouse il 10 m1000b, by R&D Systems (2 mentions) Protein transport inhibitor cocktail, by BD (2 mentions) Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (2 mentions) Pe cy7 anti mouse cd5 53 7.3, by Thermo Fisher Scientific (2 mentions) Fitc anti mouse il 10 jes5 16e3, by BioLegend (2 mentions) Mouse tnf α 560478 elisa kit, by BD (2 mentions) Macrophage colony stimulating factor m csf 300 25, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Goat serum, by Thermo Fisher Scientific (1 mentions) Rotary microtome, by Zeiss (1 mentions) He staining kit, by Beyotime (1 mentions) Paraformaldehyde solution, by Biosharp (1 mentions) Anti cd28 clone 37, by BioLegend (1 mentions) M csf 300 25, by Thermo Fisher Scientific (1 mentions) Mouse il 6 opteia 555240, by BD (1 mentions) Mouse tnf α opteia 560478, by BD (1 mentions) α minimal essential media, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)

6 protocols using acid phosphatase leukocyte trap kit 387a

Histological Evaluation of Rat Bones and DRGs

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Rats were anesthetized with overdose of sodium pentobarbital. Then rat tibiae and lumbar 4 (L4) -L5 dorsal root ganglions (DRGs) were fixed by immersing in 4% paraformaldehyde solution (Biosharp, Beijing, China). After decalcification, tibiae and DRGs were dehydrated and embedded in paraffin for cutting into 8 µm sections via the rotary microtome (Carl Zeiss, Germany).
For tibia hematoxylin-eosin staining and tartrate-resistant acid phosphatase (TRAP) staining, the sections were stained with HE staining kit (Beyotime, Shanghai, China) and Acid phosphatase, Leukocyte (TRAP) kit (387A, Sigma-Aldrich, Germany) following the manufacturer's protocol.
For the immunohistochemical staining, tibia and DRG sections were incubated with methanol containing 3% H₂O₂ for 15 min. After antigen repair, sections were blocked with 10% goat serum (Gibco) in TBS for 3 h. Then sections were incubated with primary antibody at 4°C overnight. After washing and horseradish peroxidase enzyme labeled secondary antibodies incubation, DAB substrate solution and hematoxylin was used according to the previous report (Hao et al., 2018 (link)).

Ju L., Hu P., Chen P., Wu J., Li Z., Qiu Z., Cheng J, & Huang F. (2021). Corydalis Saxicola Bunting Total Alkaloids Attenuate Walker 256-Induced Bone Pain and Osteoclastogenesis by Suppressing RANKL-Induced NF-κB and c-Fos/NFATc1 Pathways in Rats. Frontiers in Pharmacology, 11, 609119.

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Immunomodulatory Reagents for Cell Culture

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The following antibodies/kits were procured from eBiosciences (USA): APC Anti-Mouse/Rat-Foxp3 (FJK-16s) (17-5773), PE Anti-Human/Mouse-Rorγt (AFKJS-9) (12-6988), Foxp3/Transcription factor staining buffer (0-5523-00) and RBC lysis buffer (00-4300-54). PE/Cy7 Anti-Mouse CD8 (53-6.7) (100721), PE Anti-Mouse CD254-(TRANCE-RANKL) (IK22/5) (510005). Anti-IFN-γ (clone: XMG1.2), Anti-IL-4 (clone: 11B11), Anti-CD3e (clone: 145-2C11) and Anti-CD28 (clone: 37.51) antibodies were procured from BioLegend (USA). Following ELISA kits were brought from R&D: Mouse IL-10 (M1000B) and Mouse IL-17 (M1700) Quantikine ELISA kits. The Following ELISA kits and reagents were brought from BD (USA): Mouse IL-6 (OptEIA-555240), Mouse TNF-α (OptEIA-560478). Acid phosphatase, leukocyte (TRAP) kit (387A), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), FITC-Phalloidin (P5282) and DAPI were bought from Sigma (USA). Macrophage-colony stimulating factor (M-CSF) (300-25) and Receptor activator of nuclear factor κB-ligand (sRANKL) (310-01), Human TGF-β1 (AF-100-21C), Murine IL-2 (AF-212-12), Murine IL-6 (AF-216-16), Murine IL-23 (200-23), were procured from PeproTech (USA). MRS broth (GM369) was purchased from HiMedia (India). α-Minimal essential media and RPMI-1640 were purchased from Gibco (USA).

Sapra L., Dar H.Y., Bhardwaj A., Pandey A., Kumari S., Azam Z., Upmanyu V., Anwar A., Shukla P., Mishra P.K., Saini C., Verma B, & Srivastava R.K. (2021). Lactobacillus rhamnosus attenuates bone loss and maintains bone health by skewing Treg-Th17 cell balance in Ovx mice. Scientific Reports, 11, 1807.

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Murine Immune Cell Identification

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The following antibodies/kits were obtained from eBiosciences (USA): PerCp-Cy5.5 Anti-Mouse-CD19 (1D3) (45-0193-82), PE-Cy7 Anti-Mouse-CD5 (53-7.3) (25-0051-81), APC Anti-Mouse-CD1d (1B1) (17-0011-82), Foxp3/Transcription factor staining buffer (0-5523-00), and RBC lysis buffer (00-4300-54). FITC Anti-Mouse-IL-10 (JES5-16E3) (505005) was purchased from Biolegend (USA). InVivoMAb anti-mouse IL-10 antibody (BE0049) and InVivoMAb Rat IgG1 isotype control were purchased from BioXcell (USA). Protein transport inhibitor cocktail and the Mouse TNF-α (560478) ELISA kit were procured from BD (USA). The following ELISA kits were brought from R&D and Elabscience: Mouse IL-10 (M1000B) (E-EL-M0046) and Mouse IL-17 (M1700). PMA, Ionomycin, LPS (Escherichia coli serotype 0111: B4), FITC-Phalloidin (P5282), DAPI, and Acid phosphatase leukocyte (TRAP) kit (387A) were procured from Sigma-Aldrich (USA). Macrophage-colony stimulating factor (M-CSF) (300-25) and Receptor activator of nuclear factor κB-ligand (sRANKL) (310-01) were procured from PeproTech (USA). α-Minimal essential media (MEM) and RPMI-1640 were obtained from Gibco (Thermo Fisher Scientific, USA).

Sapra L., Bhardwaj A., Mishra P.K., Garg B., Verma B., Mishra G.C, & Srivastava R.K. (2021). Regulatory B Cells (Bregs) Inhibit Osteoclastogenesis and Play a Potential Role in Ameliorating Ovariectomy-Induced Bone Loss. Frontiers in Immunology, 12, 691081.

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Osteoclast Formation via TRAP Staining

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After induction of culture for 7 days, TRAP staining was performed to evaluate TRAP(+)-multinucleated osteoclast formation. RAW 264.7 cells were cultured on a 24-well plate at a density of 2×10⁴ cells/well and allowed to adhere overnight. DMEM was then replaced, and the cells were treated with new DMEM containing 100 ng/ml RANKL. After induced culture for 7 days, the cells were stained for TRAP expression using an Acid Phosphatase, Leukocyte (TRAP) kit (387-A; Sigma-Aldrich) following the manufacturer's instructions.

Feng Y.L., Jiang X.T., Ma F.F., Han J, & Tang X.L. (2017). Resveratrol prevents osteoporosis by upregulating FoxO1 transcriptional activity. International Journal of Molecular Medicine, 41(1), 202-212.

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Osteoclast Identification and Quantification

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The cells were stained using an Acid Phosphatase, Leukocyte TRAP kit (387-A; Sigma-Aldrich, St. Louis, MO, USA) to confirm the generation of TRAP-positive cells, which appeared dark red. TRAP-positive multinucleated cells containing three or more nuclei were considered to be osteoclasts. The osteoclasts were then counted and images were captured using a microscope (microscope, JEM1200EX; Camera, Olympus E330; Olympus Corporation, Tokyo, Japan).

XU J., YUE F., WANG J., CHEN L, & QI W. (2014). High glucose inhibits receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via downregulation of v-ATPase V0 subunit d2 and dendritic cell-specific transmembrane protein. Molecular Medicine Reports, 11(2), 865-870.

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Immunological Profiling of Mouse Cells

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The following antibodies/kits were obtained from eBiosciences (USA): PerCp-Cy5.5 Anti-Mouse-CD19 (1D3) (45-0193-82), PE-Cy7 Anti-Mouse-CD5 (53-7.3) (25-0051-81), APC Anti-Mouse (CD1d) (1B1) (17-0011-82), Foxp3/Transcription factor staining buffer (0-5523-00) and RBC lysis buffer (00-4300-54). FITC Anti-Mouse-IL-10 (JES5-16E3) (505005) was purchased from Biolegend (USA). Protein transport inhibitor cocktail and Mouse TNF-α (560478) ELISA kit were procured from BD (USA). The following ELISA kits were brought from R&D: Mouse IL-10 (M1000B) and Mouse IL-17 (M1700). PMA, Ionomycin, LPS (Escherichia coli serotype 0111: B4) and Acid phosphatase leukocyte (TRAP) kit (387A) were procured from Sigma-Aldrich (USA). Macrophage-colony stimulating factor (M-CSF) (300-25) and Receptor activator of nuclear factor κB-ligand (sRANKL) (310-01) were procured from peprotech (USA). α-Minimal essential media (MEM) and RPMI-1640 were obtained from Gibco (Thermo Fisher Scientific, USA).

Sapra L., Bhardwaj A., Mishra P.K., Verma B., & Srivastava R.K. (2021). Regulatory B cells (Bregs) inhibit osteoclastogenesis and prevent bone loss in osteoporotic mice model.

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