A Modified Simple Method for Induction of Myocardial Infarction in Mice

  • Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging and require commercially available apparatus. The current study describes a simple and efficient process for induction of MI in mice with readily available materials.
  • The mouse chest skin was cut open under stable anesthesia with a simplified anesthesia device made of centrifuge tubes. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an Corning Centrifuge Tubes MI model can be established in mice within 2-3 min with readily available materials.

Extending the working properties of liquid platelet-rich fibrin using chemically modified PET tubes and the Bio-Cool device

 Centrifuge Tube
Centrifuge Tube
  1. Platelet-rich fibrin (PRF) has been utilized in regenerative medicine as a concentration of autologous platelets and growth factors that stimulates tissue regeneration. More recently, liquid-PRF (also called injectable-PRF; i-PRF) has been brought to market utilizing PET plastic tubes. Due to new advances made in tube technology, the first aim of the present study was to investigate the liquid consistency of liquid-PRF utilizing both standard and chemically modified PET plastic tubes. Furthermore, it is well known that the conversion of PRF into a fibrin matrix is derived from the temperature-controlled enzymatic process that converts liquid fibrinogen and thrombin to solid fibrin. This study also investigated for the first time the use of a cooling device (Bio-Cool) to extend the liquid working properties of liquid-PRF.
  2. In total, 30 participants enrolled in this study. From each patient, four tubes of liquid-PRF were drawn, two standard white Vacuette tubes and two blue chemically modified hydrophobic tubes. Following centrifugation at 700 RCF-max for 8 min in a Bio-PRF horizontal centrifuge, one white and one blue tube were kept upright at room temperature, while the other white and blue tube were placed within the cooling device. Thereafter, the liquid-PRF layers were monitored over time until clotting occurred. Patient gender, age, and altitude above sea level (+ 5000 ft) were recorded and compared for clotting times.
  3. The findings from the present study demonstrated that the chemically modified PET tubes performed 37% better than the control tubes (extended the working properties of liquid-PRF by over 20 min). Most surprisingly, tubes kept in the cooling device demonstrated an average of 90 min greater working time (270% improvement). While patients living at altitude did significantly improve the clotting ability of liquid-PRF, no differences were observed when comparing male vs female or younger vs older patients in liquid-PRF clotting times.
  4. Cooling of blood following centrifugation represented a 270% improvement in working properties of liquid-PRF. Optimization of liquid-PRF tubes utilizing chemically modified hydrophobic PET tubes also delayed the clotting process by 37%. Patient gender and age had little relevance on liquid-PRF.
  5. The present findings demonstrate for the first time that cooling of liquid-PRF is able to extend the working properties of liquid-PRF by over 90 min. Thus for clinicians performing longer clinical procedures, the cooling of blood may represent a viable strategy to improve the working time of liquid-PRF in clinical practice. Bio-PRF; Cooling device; PRF; Platelet-rich fibrin; Wound healing.

Fixation and Immunostaining of Endogenous Proteins or Post-translational Modificationsin Caenorhabditis elegant.

  • Although the advent of genetically-encoded fluorescent markers, such as the green fluorescent protein (GFP; Chalfie et al., 1994 ), has enabled convenient visualization of gene expression in vivo, this method is generally not effective for detecting post-translational modifications because they are not translated from DNA sequences.
  • Genetically-encoded, fluorescently-tagged transgene products can also be misleading for observing expression patterns because transgenes may lack endogenous regulatory DNA elements needed for precise regulation of expression that could result in over or under expression. Fluorescently-tagged proteins created by CRISPR genome editing are less prone to defective expression patterns because the loci retain endogenous DNA elements that regulate their transcription (Nance and Frøkjær-Jensen, 2019).
  • However, even CRISPR alleles encoding heritable fluorescently-tagged protein markers can result in defects in function or localization of the gene product if the fluorescent tag obstructs or otherwise interferes with important protein interaction domains or affects the protein structure. Indirect immunofluorescence is a method for detecting endogenous gene expression or post-translational modifications without the need for transgenesis or genome editing.
  • Here, we present a reliable protocol in which C. elegans nematodes are fixed, preserved, and permeabilized for staining with a primary antibody to bind proteins or post-translational modifications, which are then labeled with a secondary antibody conjugated to a fluorescent dye.
  • Use of this method may be limited by the availability of (or ability to generate) a primary antibody that binds the epitope of interest in fixed animals. Thousands of animals are simultaneously subjected to a series of chemical treatments and washes in a single centrifuge tube, allowing large numbers of identically-treated stained animals to be examined.
  • We have successfully used this protocol (O’ Hagan et al., 2011 and 2017; Power et al., 2020 ) to preserve and detect post-translational modifications of tubulin in C. elegans ciliated sensory neurons and to detect non-modified endogenous protein (Topalidou and Chalfie, 2011).

Transfer some Hydrogel Containing 5-Fluorouracil and Etodolac Combination for Synergistic Oral Cancer Treatment

  • Oral cancer is one of the most common malignancies with an increased rate of incidence. 5-Fluorouracil (5FU) is an effective chemotherapeutic indicated for oral cancer treatment. Etodolac (Et), a cyclooxygenase-2 inhibitor, can be used as an adjuvant agent to sensitize cancer cells to chemotherapy. The aim of this work was to prepare and characterize 5FU and Et dual drug-loaded transfersomes to treat oral cancer.
  • Transfersomes were prepared by thin-film hydration method and characterized for the average particle size and zeta-potential using dynamic light scattering and scanning electron microscopy techniques.
  • The prepared transfersomes were further characterized for their drug loading, entrapment efficiencies using amicon centrifuge tubes and drug release behavior using cellulose membrane. The synergistic activity of dual drug-loaded transfersomes was studied in FaDu oral cancer cells. Results showed that the average particle size, polydispersity index, and zeta potential were 91±6.4 nm, 0.28±0.03, and (-)46.9±9.5 mV, respectively, for 5FU- and Et (1:1)-loaded transfersomes.

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R006-G Bio Basic 1 UNIT

5-Well Centrifuge Tube Box (10cc Glass Vials)

R007-G Bio Basic 1 UNIT

Centrifuge adapter pack for 7ml tube

HOM1028 Scientific Laboratory Supplies EACH

CoolRack for 1 x 250ml conical centrifuge tube

432063 Scientific Laboratory Supplies EACH

Greiner 50ml Centrifuge tube conical base no cap

G227201 Scientific Laboratory Supplies PK500

Block for 4pcs x 15mL Centrifuge Tube, WSC-2610

4002645 Atto 2unit

Block for 2pcs x 50mL Centrifuge Tube, WSC-2610

4002646 Atto 2unit

Block for 4pcs x 15mL Centrifuge Tube, WSC-2610

4002645-2 Atto each

Axygen Micro Centrifuge Tube Maxyclear 5mL Sterile

AXY2178 Scientific Laboratory Supplies PK1250

PCR STRIP TUBE ROTOR FOR MINI CENTRIFUGE

6770-RTS CORNING 1/pk

Greiner 50ml Centrifuge tube skirted racked sterile

G210270 Scientific Laboratory Supplies PK300

Block for 24 pcs x 5mL Centrifuge Tube, WSC-2620/30

4002660 Atto 1unit

Block for 24pcs x φ12mm Centrifuge Tube, WSC-2620/30

4002655 Atto 1unit

Block for 12pcs x 15mL Centrifuge Tube, WSC-2620/30

4002656 Atto 1unit

Conical Centrifuge Tube, PP/HDPE, 15ml, RCF 13000xg, 500/case

SP61126 Bio Basic 1CS, 500UNIT

Conical Centrifuge Tube, PP/HDPE, 50ml, RCF 14000Xg, 500/case

SP61161 Bio Basic 1CS, 500UNIT

Block for 6 pcs x 50mL Centrifuge Tube, WSC-2620/30

4002657 Atto 1unit

Greiner 15ml Centrifuge tube graduated racked sterile

G188261 Scientific Laboratory Supplies PK500

Scienceware centrifuge tube rack epoxy-coated steel

R8384-1EA Scientific Laboratory Supplies EACH

Scienceware centrifuge tube rack epoxy-coated steel

R8509-1EA Scientific Laboratory Supplies EACH

Scienceware centrifuge tube rack epoxy-coated steel

R8634-1EA Scientific Laboratory Supplies EACH
The highest encapsulation efficiency achieved was 36.9±3.8% and 79.8±6.4% for 5FU and Et (1:1), respectively. Growth inhibition studies in FaDu cells using different concentrations of 5FU and Et showed a combination index of 0.36, indicating a synergistic effect. The FaDu cell uptake of drug-loaded transfersomes was significantly (p<0.05) greater than that of free drugs. The transfersome hydrogel made of HPMC (2% w/w) showed similar flux, lag time, and permeation coefficient as that of drug-loaded transfersomes across excised porcine buccal tissue. In conclusion, 5FU and Et transfersome hydrogel can be developed for localized delivery to treat oral cancer.

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