29 gauge needle

With the chest open, synthetic oligonucleotides pretreated with 15 µl Dharmafect Duo (Thermo) were injected into the myocardium through an insulin syringe with a 29-gauge needle (BD). The dosages used per mouse were 80 ng miR-22 mimic, 1 nmol Mecp2 siRNAs On-TARGETplus SMART pool (a pool of four target-specific 20–25 nt siRNAs; Thermo). One µg exosomes (Exo^non-IPC, Exo^IPC, Exo^{IPC+miR-22 Inhibitor}) were injected along the border between infarct zone and normal myocardium after LAD.

A U-100 insulin syringe equipped with a 29-gauge needle (BD) was used to inject 10 μL aliquots of a 1 × 10⁶ cfu mL⁻¹ bacterial inoculum in PBS or 10 mM MgCl₂ into the haemocoel of each G. mellonella larvae via the last left proleg. Before and after injection, the area was cleaned using an alcohol swab. After injection, G. mellonella larvae were incubated in plastic containers and kept at room temperature in the dark. The number of dead larvae were scored daily for 72 h. Larvae were considered dead when they displayed no movement in response to a stab to the head. The crude cell-free supernatant (derived by centrifuging and filtering (0.2 μM)) from an overnight (O/N) broth culture of the bacterial strains was also used to assess toxicity.

For in vivo implantation, cells were divided into six groups, 1 × 10⁶ mouse dermal cells alone used as a control group (n =8), and 5 × 10⁵ P1-HFSCs mixed with 5 × 10⁵ mouse dermal cells, which were used in five different experimental groups (n =8 per group): HFSCs, LbL-coated HFSCs (LbL-HFSCs), HFSCs + TGF-β2 (10 pM), LbL-coated HFSCs loaded with TGF-β2 [LbL(TGF-β2)-HFSCs], and 50% LbL-HFSCs + 50% LbL (TGF-β2)-HFSCs (50% + 50%). Athymic nude mice were anesthetized with pentobarbital sodium (1.3 mg/kg body weight). Next, 1 × 10⁶ cells from each group were injected subcutaneously into the dorsal side in a total volume of 50 µL PBS using a 29-gauge needle (BD Biosciences). After 3 wk, the grafted specimens were examined under a stereomicroscope (MVX10, Olympus) and photographed. The grafted regions were harvested, fixed in 4% paraformaldehyde, and embedded in paraffin. Subsequently, 4-µm thick serial sections were stained with hematoxylin and eosin (H&E). Representative areas for each group were selected and photographed for further analysis.

Leishmania infantum (MCAN/BR/00/BA262) promastigotes isolated from a naturally infected dog (Bahia State, Brazil) were cultured in Schneider’s medium (LGC, Brazil) supplemented with 10% of inactivated FBS (fetal bovine serum) (Gibco, USA), 2 mM L-glutamine, 100 IU/ml penicillin, 1% streptomycin (Gibco, USA).
Fifteen days after the last immunization, hamsters were inoculated by intradermal route, in the left ear with 105 stationary phase promastigotes plus 0.5 pair of sonicated salivary glands (SGH) from female Lutzomyia longipalpis, using a 29-gauge needle (BD Ultra-Fine) in 20uL of saline. Salivary glands were dissected from 5- to 7-day-old females and stored in endotoxin-free PBS at −70°C. Salivary glands were sonicated (Sonifer 450 homogenizer, Branson, Danbury, Connecticut), and afterwards centrifuged at 12,000 g for 5 minutes. Supernatant was collected and used immediately.

Six to eight-week-old female hamsters were immunized with 10⁶ total stationary-phase LmexCen^−/− parasites by intradermal injection in the left ear in 10 μl PBS using a 29-gauge needle (BD Ultra-Fine). In addition, the age-matched control group of animals were infected with 10⁶ total stationary-phase L. mexicana wild-type (LmexWT) promastigotes. Lesion size was monitored weekly by measuring the diameter of the ear lesion using a direct reading vernier calliper. Parasite burdens in the ear, draining lymph node (dLN), spleen, liver and bone marrow tissues were determined by limiting dilution assay^{27 (link)}. Ear tissues were treated with Liberase enzyme (Sigma-Aldrich) (0.15 mg/ml) in DMEM mediun at 37 °C. After 90 min of incubation tissues were grinded to make single cell suspensison using BD medimachine system. Cells were washed two times with M199 medium. Lymph nodes, spleen, liver and bone marrow were harvested and homogenized with a cell strainer in 2 ml of M199 medium supplemented with 10% FBS and 1% Penicillin/Streptomycin. To determine parasite burden cell suspension from each organs were serially diluted in 96 well tissue culture plates. After 12 days of incubation at 26 °C, plates were examined with a inverted microscope and parasite burden were calculated for each organ.

Cells were subjected to three washes with PBS and subsequently lysed using SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, 10% glycerol, and 0.025% bromophenol blue). High-molecular-weight DNA was sheared by passing the lysate through a 29-gauge needle (326,666, BD Biosciences). After centrifugation at 15,000 rpm for 5 min and heat treatment at 55 °C for 20 min, the samples were electrophoresed. Detection was performed using horseradish peroxidase (HRP)-conjugated secondary mouse or rabbit antibodies and chemiluminescence HRP substrate (WBKLS0100, Millipore-Sigma), and the signals were captured using an image analyzer (LAS500, GE Healthcare, Chicago, IL, USA).