To identify the osteogenic potential within the synthetic BIM, immunohistochemistry staining for osteocalcin, tartrate-resistant acid phosphatase (TRAP) and alizarin red staining were carried out on the 7 μm-thick sections. TRAP staining of osteoclasts was performed using Leukocyte acid phosphate kit (Sigma) with fast red violet according to the manufacture’s instruction. Immunohistochemistry staining of osteoclacin and alizarin red staining were preformed as described above.
Acid phosphate leukocyte kit
Manufactured by Merck Group
Sourced in United States
The Acid Phosphate Leukocyte Kit is a laboratory equipment product designed to assist in the detection and quantification of acid phosphatase levels in leukocytes. It provides a standardized method for the analysis of this enzyme, which is commonly associated with various hematological and immunological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Thermo Fisher Scientific (4 mentions)
M csf, by Thermo Fisher Scientific (4 mentions)
Rankl, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Leica dm2500, by Leica camera (3 mentions)
Osteomeasure, by OsteoMetrics (1 mentions)
Ventana discovery ultra, by Roche (1 mentions)
11 protocols using acid phosphate leukocyte kit
1
Evaluating Osteogenic Potential of Synthetic BIM
2
Histological Analysis of Bone and Cartilage in AIA Mice
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Bone tissues were fixed in 4% formaldehyde, decalcified in 10% EDTA, paraffin‐embedded, and cut into 20‐μm thick sections (sectioning was performed at Histocenter, Mölndal, Sweden). The knee sections from AIA mice were stained with hematoxylin and eosin. For osteoclast staining, sections were stained for tartrate resistant acid phosphatase (TRAP) using a Leukocyte Acid Phosphate kit (Sigma‐Aldrich). Synovitis, bone erosion, and cartilage damage were graded in a blinded manner (by researchers CE and ES) using a blunt three‐grade histological scoring system (mild, moderate, or severe) as described by Liphardt and colleagues.(33 )Osteoclast number and osteoclast surface attached to the bone were quantified in the epiphyseal and metaphyseal parts of the tibia in the knee sections from the AIA experiment, and both osteoclast and osteoblast number and surface attached to the bone were quantified in the metaphyseal part of the femur in the 6‐week‐old and 16‐week‐old female mice. Osteoclasts were defined as TRAP‐positive cells with three or more nuclei and cuboidal cells at the bone surface were considered as osteoblasts. All histological analyses were performed using a microscope (Nikon, Tokyo, Japan) and the image analysis system Osteomeasure (OsteoMetrics, Decatur, GA, USA). All histological assessments were performed in a blinded manner.
3
Detecting Osteoclast Differentiation via TRAcP Staining
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Plates for detecting tartrate resistant acid phosphatase (TRAcP) activity staining were harvested in duplicate on t = 21. Cells were washed with PBS and fixed with 4% PBS buffered formaldehyde for 10 min before being stored with PBS at 4°C. After washing the cells with water at 37°C, TRAcP staining solution was made consisting of Fast Garnet GBC base, sodium nitrate, acetate, naphthol AS-BI and tartrate in AD on 37°C, using the leukocyte acid phosphate kit (Sigma) following the manufacturer’s instructions. The cells were counterstained with DAPI (diamidino-2phenylindole dihydrochloride). A distinction was made between cells with 3-5 nuclei, and cells with ≤6 nuclei. For analyses, the average of the number counted in the duplicate wells per patient was used.
4
Bone Tissue Fixation and Staining
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Bone tissue samples were fixed in 4% paraformaldehyde overnight and decalcified in 10% EDTA for 10 days. Tissues were paraffin-embedded, and 7-µm sections were adhered to glass slides. Anti-GFP IHC was performed with Ventana Discovery Ultra (Tucson, AR). TRAP staining was performed using a leukocyte acid phosphate kit (Sigma) with fast red violet.
5
Quantifying Osteoclast Formation and Bone Resorption
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OCs were stained for tartrate-resistant acid phosphatase (TRAP) at days 7, 14, and 21 using the Acid Phosphate Leukocyte Kit (Sigma-Aldrich) according to the manufacturer's instructions. Multinuclear cells containing three or more nuclei [16 (link), 17 (link)] were counted as TRAP positive OCs. After visualization, cells were removed from bone slices using sodium hypochlorite and stained with 0.1% toluidine blue for the measurement of resorbed area at days 7, 14, and 21 of culture [18 (link)]. Bone slices were photographed in an area of 1.25 mm2 with a bright field microscope (Leica DM2500, Leica). The number of TRAP stained OCs was counted at each time point and resorption pits were traced using ImageJ software (NIH, Bethesda, MD). The resorbed area was expressed in % of total area.
6
Monocyte-Derived Osteoclast Differentiation
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Purified human CD14+ monocytes were seeded at 2.5 × 105 cells/well in 96-well plates containing α-minimum essential medium (α-MEM) with fetal bovine serum (FBS) (10%, v/v; Invitrogen, Waltham, MA, USA) and M-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) for 3 days. RANKL (25 ng/mL) and TNF-α (50 ng/mL) (both from PeproTech, Rocky Hill, NJ, USA) were added every 3 days for 9 days to induce osteoclast differentiation. The osteoclasts were identified by staining with tartrate-resistant acid phosphatase (TRAP) on Day 13 using the Acid Phosphate Leukocyte Kit (Sigma, St. Louis, MO, USA). TRAP-stained cells containing three or more nuclei were defined as osteoclasts. [20 (link)] The numbers of osteoclasts were counted and averaged from four high-power fields (HPFs) (100×) per well.
7
Quantifying Osteoclast Formation and Bone Resorption
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Tartrate-resistant acid phosphatase (TRAP) staining of OCs was performed at days 7, 14 and 21 of culture using the Acid Phosphate Leukocyte Kit (TRAP, Sigma-Aldrich) according to the manufacturer's instructions. OCs were counted as TRAP positive cells containing three or more nuclei [14 (link),15 (link)]. For measurement of resorbed area in the resorption assay at days 7, 14 and 21 of culture, cells were removed from the bone slices using sodium hypochlorite and stained with 0.1% toluidine blue [16 (link)]. Bone slices from both TRAP staining and resorption functional assays were photographed in an area of 1.25 mm2 with a brightfield microscope (Leica DM2500, Leica) under a 10x objective. The number of TRAP stained OCs was counted for each time-point per condition and the resorption pits were traced using ImageJ software (NIH, Bethesda, MD). The resorbed area was calculated and expressed in % of total area.
8
Generation and Analysis of Osteoclasts
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Then, 3 × 105 purified human CD14+ monocytes were seeded in 24-well plates containing a-MEM with FBS (10%, v/v; Invitrogen, Waltham, MA, USA) and M-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) for three days. RANKL (100 ng/mL; PeproTech, Rocky Hill, NJ, USA) and TNF-α (100 ng/mL; PeproTech, Rocky Hill, NJ, USA) were added to induce osteoclast differentiation [11 (link)]. The osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP) at day 13 using the Acid Phosphate Leukocyte Kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. TRAP-stained cells containing three or more nuclei were defined as osteoclasts [30 (link)]. We measured the number of osteoclasts from four randomly selected higher power fields (HPF) (200×) per quadrant of the well. Four HPF were chosen from each quadrant. The number of osteoclasts was counted from the average value from 16 HPFs.
9
Osteoclast Differentiation from Monocytes
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Purified human CD14+ monocytes were seeded at a density of 3 × 105 cells/well onto 96-well plates containing α-MEM with FBS (10%, Invitrogen, Waltham, MA, USA) and M-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) for 3 days. RANKL (100 ng/mL; PeproTech, Rocky Hill, NJ, USA) and TNF-α (100 ng/mL; PeproTech, Rocky Hill, NJ, USA) were added to induce osteoclast differentiation. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP) on day 13 using the Acid Phosphate Leukocyte Kit (Sigma, St. Louis, MO, USA), according to the manufacturer’s instructions. TRAP-stained cells containing three or more nuclei were defined as osteoclasts [28 (link)]. The number of osteoclasts was counted from four high power field (HPF; 100×) images per well; then, average was calculated.
10
Quantifying Osteoclast Activity: TRAP Staining and Resorption Assay
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TRAP staining of OC was performed using the Acid Phosphate, Leukocyte Kit (tartrate resistant acid phosphatase (TRAP), Sigma-Aldrich) according to the manufacturer's instructions. This assay allows the identification of mature OC (TRAP-positive cells containing three or more nuclei). In the resorption assay, to measure the resorbed area, OC were removed from the bone slices using sodium hypochlorite and stained with 0.1% toluidine blue.
Bone slices from both TRAP staining and resorption functional assays were photographed in an area of 1.25 mm2 with a brightfield microscope (Leica DM2500, Leica, Wetzlar, Germany) under a 10x objective. The number of TRAP-stained OC was counted for each time-point per condition and the resorption pits were traced using ImageJ (NIH, Bethesda, Maryland, USA), a mean value of the resorbed area was calculated and expressed in percentage of total area.
Bone slices from both TRAP staining and resorption functional assays were photographed in an area of 1.25 mm2 with a brightfield microscope (Leica DM2500, Leica, Wetzlar, Germany) under a 10x objective. The number of TRAP-stained OC was counted for each time-point per condition and the resorption pits were traced using ImageJ (NIH, Bethesda, Maryland, USA), a mean value of the resorbed area was calculated and expressed in percentage of total area.
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