Tachyzoites of the T. gondii RH strain was maintained as described previously [19 (link)]. Briefly, human retinal pigment epithelial (ARPE-19) cells (ATCC) were cultured in a 1:1 (v/v) mixture of DMEM/F12 supplemented with 10% heat-inactivated FBS and an antibiotic-antimycotic solution. ARPE-19 cells were infected with T. gondii at a multiplicity of infection (MOI) of 5 for 2–3 days. After spontaneous host cell rupture, parasites and cellular debris were pelleted by centrifugation and washed in cold PBS. The final pellet was resuspended and passed through a 26-gauge needle fitted with a filter with 5.0-μm pores (Millipore, Billerica, Massachusetts, USA).
26 gauge needle
Manufactured by Merck Group
Sourced in United States
The 26-gauge needle is a medical device used for various procedures. It is a thin, hollow needle with an outer diameter of 0.47 millimeters. The needle is designed to provide a precise and minimally invasive method for administering medications, collecting samples, or performing other medical interventions.
Automatically generated - may contain errors
Lab products found in correlation
Percoll solution, by GE Healthcare (2 mentions)
5.0 μm pore filter, by Merck Group (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
F 12 dmem f12, by Thermo Fisher Scientific (1 mentions)
Arpe 19, by American Type Culture Collection (1 mentions)
Arpe 19, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Welgene (1 mentions)
Gibco antibiotic antimycotic 100x, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Welgene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Arpe 19 cell, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
6 protocols using 26 gauge needle
1
Purification of T. gondii Tachyzoites
2
Isolation and Purification of N. caninum Tachyzoites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tachyzoites of N. caninum-1 strain (ATCC 50843) were restored at our laboratory. Vero cells were infected with tachyzoites of Nc-1 and cultured at 37°C and 5% CO2 for 3–5 days in DMEM/F12 supplemented with 2% heat-inactivated FBS and antibiotic–antimycotic reagents. After spontaneous host cell rupture, parasites and cell debris were washed in cold DMEM/F12 without FBS and harvested by centrifugation at 850 × g at 4°C for 10 min. After centrifugation, the pellet was resuspended in cold DMEM/F12 and passed through a 26-gauge needle (Millipore, Billerica, MA, United States) to further break the cells. The obtained mixture was slowly added onto 40% percoll solution (GE Healthcare, United States) in DMEM/F12 without FBS and separated by centrifugation at 850 × g in a horizontal centrifuge for 30 min. The fraction containing tachyzoites at the bottom of the tube was collected, resuspended in DMEM/F12 without FBS and centrifuged at 850 × g at 4°C for 10 min. The final pellet was purified tachyzoites.
3
Isolation of Neospora caninum Tachyzoites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tachyzoites of N. caninum-1 strain were stored at our laboratory. Vero cells were cultured in DMEM supplemented with 10% FBS. Cells were infected with Nc-1 tachyzoites and cultured in DMEM with 2% FBS for 3–5 days at 37°C and 5% CO2. After spontaneous cell rupture, cell debris mixed with tachyzoites was harvested. After centrifugation, the pellet was resuspended in cold DMEM medium and passed through a 26-gauge needle (Millipore, Billerica, MA, United States). The obtained mixture was slowly layered on a 40% Percoll solution (GE Healthcare, United States) in DMEM without FBS and separated by centrifugation at 850 ×g in a horizontal centrifuge for 30 min. The fraction containing tachyzoites at the bottom of the tube was collected and washed in DMEM without FBS by centrifugation at 850 ×g at 4°C for 10 min. The final tachyzoite pellet was resuspended in DMEM without FBS.
4
Purification of Tachyzoites from T. gondii RH Strain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tachyzoites of T. gondii RH strain were maintained as described previously, with some modifications [24 (link)]. Briefly, human retinal pigment epithelial cells, ARPE-19 (ATCC, Manassas, Virginia, USA), were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) with F12 (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotic–antimycotics (all from Gibco, Grand Island, New York, USA).
ARPE-19 cells were infected with RH strain of T. gondii using a multiplicity of infection (MOI) of 5 and incubated at 37°C and 5% CO2 for 2–3 days. Following spontaneous host cell rupture, parasites and host cell debris were washed in cold PBS. The final pellet was resuspended in cold DMEM and then passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, Massachusetts, USA).
ARPE-19 cells were infected with RH strain of T. gondii using a multiplicity of infection (MOI) of 5 and incubated at 37°C and 5% CO2 for 2–3 days. Following spontaneous host cell rupture, parasites and host cell debris were washed in cold PBS. The final pellet was resuspended in cold DMEM and then passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, Massachusetts, USA).
5
Purification of GFP-expressing Toxoplasma gondii
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T. gondii RH strain was multiplied in ARPE-19 cells at a multiplicity of infection (MOI) of 5 and grown for 2–3 days at 37°C and 5% CO2. T. gondii RH strain expressing transgenic green fluorescent protein (GFP-RH) were kindly provided by Dr. Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan). Host cell debris and parasites were washed in phosphate-buffered saline (PBS) after spontaneous host cell rupture. Final pellet was suspended in cold DMEM, and then passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, Massachusetts, USA).
6
Differentiation and Infection of Macrophages and RPE Cells with Toxoplasma gondii
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were differentiated over 5–7 days in a medium with a recombinant macrophage colony-stimulating factor (M-CSF), as described previously [42 (link)]. The culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Waltham, MA, USA) and 1% antibiotic–antimycotic (Gibco™ antibiotic–antimycotic (100X); Gibco BRL, Waltham, MA, USA). Human retinal pigment epithelial ARPE-19 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM/F-12 (Welgene, Gyeongsan, Korea) with 10% FBS and 1% antibiotic–antimycotic.
The T. gondii RH strain was grown in ARPE-19 cells (MOI = 5) for 2–3 days at 37 °C and 5% CO2. Host cells and parasites were washed with phosphate-buffered saline (PBS) after spontaneous host cell disruption. Protozoans were suspended in cold medium and passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, MA, USA). The GFP-RH strain was kindly provided by Dr. Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan). The T. gondii ME49 strain was obtained from the brain tissue of BALB/c mice that infected 50 cysts and were maintained every 3 weeks.
The T. gondii RH strain was grown in ARPE-19 cells (MOI = 5) for 2–3 days at 37 °C and 5% CO2. Host cells and parasites were washed with phosphate-buffered saline (PBS) after spontaneous host cell disruption. Protozoans were suspended in cold medium and passed through a 26-gauge needle and a 5.0 μm pore filter (Millipore, Billerica, MA, USA). The GFP-RH strain was kindly provided by Dr. Yoshifumi Nishikawa (Obihiro University of Agriculture and Veterinary Medicine, Japan). The T. gondii ME49 strain was obtained from the brain tissue of BALB/c mice that infected 50 cysts and were maintained every 3 weeks.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!