PMID- 31890980
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20220412
IS  - 2405-8440 (Print)
IS  - 2405-8440 (Electronic)
IS  - 2405-8440 (Linking)
VI  - 5
IP  - 12
DP  - 2019 Dec
TI  - Lipophilic tracer Dil and fluorescence labeling of acridine orange used for 
      Leishmania major tracing in the fibroblast cells.
PG  - e03073
LID - 10.1016/j.heliyon.2019.e03073 [doi]
LID - e03073
AB  - BACKGROUND: This study aims to evaluate the use of fluorescent dye Dil and super 
      vital dye acridine orange (AO) in vitro tracking of labeled L. major in the 
      fibroblast cells. METHODS: Dil crystal and AO were used to stain L. major in a 
      co-culture of the fibroblasts with the parasite. AO staining solution was added 
      to 1 x 10(6) parasites. After 10 min, the stained parasites were centrifuged and 
      washed seven times with phosphate buffered saline (PBS). The stained promastigote 
      was incubated with fibroblasts for 6-8 h. The presence of stained parasites with 
      AO in the fibroblast was assessed using a fluorescence microscope. 1 x 10(6)/mL 
      promastigote of L. major was gently suspended and mixed by Dil staining solution 
      with an ultimate concentration of 0.002 mug/mL and it was kept for 20 min at the 
      room temperature. Subsequently, after washing it in PBS for seven times, it was 
      centrifuged at 3000 rpm for 10 min. The supernatant was removed and the 
      precipitate containing stained promastigote was suspended in fresh DMEM F12 with 
      fibroblasts at 37  degrees C for 6 h. The presence of stained parasites with Dil in 
      fibroblast was assessed using a fluorescence microscope. Fibroblast 
      characterization was undertaken by a real-time polymerase chain reaction (PCR). 
      RESULTS: Acridine orange staining assisted in detection of the live parasite in 
      the fibroblast cells. Free promastigote looked green before entering into the 
      fibroblasts after 12 h culture. The parasite entered the cytoplasm of fibroblasts 
      at the beginning of the exposure and gradually entered the nucleus of the 
      fibroblast. The fibroblast nucleus was entirely stained green by AO. The L. major 
      appeared green under the fluorescent microscope. Dil staining revealed that the 
      internalized parasites with red/orange color were localized within the cytoplasm 
      after 6-hours and the nucleus of the fibroblasts after 72-hours following 
      culture. Human fibroblasts were positive at the expression of CD10, CD26, matrix 
      metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) and negative 
      for CD106 and integrin alpha 11. CONCLUSION: The fluorescent dye Dil staining is 
      a safe, easy to use, inexpensive and fast method for labeling of the Leishmania 
      parasite in the fibroblast cells. Acridine orange staining could be useful for 
      tracing the parasites in the fibroblasts too. In this study, both Dil and AO were 
      compared and considered as suitable vital dyes for identifying labeled Leishmania 
      in the fibroblast in vitro, but Dil was superior to AO with its feature does not 
      transfer from the labeled to unlabeled cells.
CI  - (c) 2019 Published by Elsevier Ltd.
FAU - Yektaeian, Narjes
AU  - Yektaeian N
AD  - Department of Parasitology and Mycology, School of Medicine, Shiraz University of 
      Medical Sciences, Shiraz, Iran.
FAU - Mehrabani, Davood
AU  - Mehrabani D
AD  - Stem Cells Technology Research Center, Shiraz University of Medical Sciences, 
      Shiraz, Iran.
FAU - Sepaskhah, Mozhdeh
AU  - Sepaskhah M
AD  - Molecular Dermatology Research Center, Shiraz University of Medical Sciences, 
      Shiraz, Iran.
FAU - Zare, Shahrokh
AU  - Zare S
AD  - Stem Cells Technology Research Center, Shiraz University of Medical Sciences, 
      Shiraz, Iran.
FAU - Jamhiri, Iman
AU  - Jamhiri I
AD  - Stem Cells Technology Research Center, Shiraz University of Medical Sciences, 
      Shiraz, Iran.
FAU - Hatam, Gholamreza
AU  - Hatam G
AD  - Basic Sciences in Infectious Diseases Research Center, Shiraz University of 
      Medical Sciences, Shiraz, Iran.
LA  - eng
PT  - Journal Article
DEP - 20191218
PL  - England
TA  - Heliyon
JT  - Heliyon
JID - 101672560
PMC - PMC6928280
OTO - NOTNLM
OT  - AO
OT  - Acridine orange
OT  - Dil
OT  - Fibroblast
OT  - Fluorescent dye
OT  - Immunology
OT  - In vitro
OT  - Infectious disease
OT  - Leishmania major
OT  - Microbiology
OT  - Molecular biology
EDAT- 2020/01/01 06:00
MHDA- 2020/01/01 06:01
PMCR- 2019/12/18
CRDT- 2020/01/01 06:00
PHST- 2018/12/01 00:00 [received]
PHST- 2019/09/15 00:00 [revised]
PHST- 2019/12/13 00:00 [accepted]
PHST- 2020/01/01 06:00 [entrez]
PHST- 2020/01/01 06:00 [pubmed]
PHST- 2020/01/01 06:01 [medline]
PHST- 2019/12/18 00:00 [pmc-release]
AID - S2405-8440(19)36732-5 [pii]
AID - e03073 [pii]
AID - 10.1016/j.heliyon.2019.e03073 [doi]
PST - epublish
SO  - Heliyon. 2019 Dec 18;5(12):e03073. doi: 10.1016/j.heliyon.2019.e03073. 
      eCollection 2019 Dec.