In vitro ookinete differentiation was performed as described previously (23 (link)). Briefly, 1 ml of mouse blood with 4 to 6% gametocytemia was collected via orbital sinus and immediately transferred to a 10-cm cell culture dish (Corning, catalog no. 801002) containing ookinete culture medium [RPMI 1640, 10% FCS, 100 μM XA, and 25 mM Hepes (pH 8.0)]. The cultures were incubated at 22°C for 12 to 14 hours to allow gametogenesis, gamete fertilization, and ookinete differentiation. Ookinete formation was monitored by Giemsa staining of culture smears. Ookinete conversion rate was calculated as the number of ookinetes per 100 female gametocytes. For time course analysis of ookinete differentiation, parasites from 2-, 4-, 6-, 8-, and 12-hour culture were collected. Ookinetes were purified using an ammonium-chloride-potassium (ACK) lysing method (23 (link)). Briefly, the cultured ookinetes were collected by centrifugation and transferred into ACK lysing buffer (A1049201, Thermo Fisher Scientific) on ice for 8 min. After erythrocytes lysis, the remaining ookinetes were isolated via centrifugation and washed twice with PBS. The ookinetes were examined and counted on the hemocytometer under 40× objective lens. Samples containing >80% ookinete population were used for further analysis.
10 cm cell culture dish
Manufactured by Corning
Sourced in United States
The 10-cm cell culture dish is a laboratory equipment used for culturing cells. It provides a sterile and controlled environment for the growth and maintenance of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Giemsa solution, by Merck Group (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
15 ml centrifuge tube, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Lymphocyte separation solution, by Corning (1 mentions)
8 protocols using 10 cm cell culture dish
1
Ookinete Differentiation from Mouse Gametocytes
2
Generation of Murine Myeloid-Derived Suppressor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation of MDSC in vitro was performed as previously described.25 (link) Briefly, 2.5×106 BM cells from healthy C57BL/6 mice were cultured for 4 days in a 10 cm cell culture dish (Corning FALCON) in 10 mL RPMI-1640 medium with GlutaMAX supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 1 mM Minimal Essential Medium (MEM) non-essential amino acids (all Thermo Fisher), 40 ng/mL GM-CSF and 40 ng/mL IL-6 (both PeproTech, recombinant murine proteins produced in Escherichia coli).
3
Obtaining conditioned medium from treated cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain the conditioned medium (CM) of SA- or Dox-treated A549 cells, cells were seeded at a density of 6 × 105 cells per 10-cm cell culture dish (Corning Inc., Durham, NC, USA) in complete RPMI medium at 37 °C and 5% CO2 in a humidified incubator. After 24 h of attachment, cells were treated with SA or Dox diluted in complete RPMI medium at desired concentrations for an additional 24 h. Subsequently, the SA- or Dox-treated cells were washed twice with fresh medium to eliminate residual SA or Dox and the medium was then replaced with serum-free RPMI medium. IL-6 gene expression was measured at various time points during the incubation (Fig. S6A ). After 24 h of incubation, the CM from control or SA/Dox-treated cells was harvested and centrifuged at 1000 × g for 5 min at 4 °C to remove cell debris. The supernatant was collected. IL-6 concentration in the CM was measured (Fig. S6B ) and the CM was stored at −80 °C until use.
4
Lentiviral Vector Production in HEK293T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells (1 × 106) were cultured in 10 cm cell culture dish (Corning). Around 80% confluency, 2.5 μg of pMD2.G (envelope plasmid), and 3.5 μg of psPAX2 (packaging plasmid) along with 4 μg (construct with gRNA) or 5 μg (construct with ABE/CBE/Cas9) of lentiviral vector were transfected using FuGENE-HD as per the manufacturer’s protocol. The viral supernatants were separately collected at 48 and 72 hr; and concentrated using Lenti-X Concentrator (Takara). The concentrated pellet was resuspended in 200 µl of 1×PBS, and the aliquots were stored at –80°C.
5
In vitro Ookinete Differentiation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
In vitro ookinete differentiation was performed as described previously (Gao et al,2018 ). Briefly, 1 ml mouse blood with 8–10% gametocytemia was collected via orbital sinus and immediately transferred to a 10‐cm cell culture dish (Corning, cat# 801002) containing ookinete culture medium (RPMI 1640, 25 mM HEPES, 10% FCS, 100 mM xanthurenic acid, pH 8.0) to allow gametogenesis, fertilization, and ookinete development at 22°C. For ookinete conversion analysis, samples from 12 h of culture were collected and stained by Giemsa solution (Sigma, cat# GS80). The conversion rate to retort and ookinete was calculated as the number of ookinetes (stages I–V) per 100 female gametocytes. The conversion rate to mature ookinete was calculated as the number of mature ookinetes (stage V) over that of total ookinetes (stages I–V). For time‐course analysis of ookinete differentiation, samples from 2, 4, 6, 8, and 12 h of culture were collected, and ookinetes in stages I and II were counted. Ookinetes were purified using ACK lysing method as described previously (Gao et al, 2018 ). Briefly, erythrocytes were lysed using the ACK lysis buffer (Thermo Fisher Scientific, A1049201), and ookinetes were counted under the hemocytometer. Purified ookinetes were used for further biochemical analysis.
+ Expand
In vitro ookinete differentiation was performed as described previously (Gao et al,
6
Transfection of ACE2-Fc Variants in HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human HEK293 cells were cultured in DMEM medium (Thermo Fisher) with 10% FBS (ATCC Manassas, VA) in a CO2 incubator at 37°C. For maintenance passage, cells were split at 1:10 twice a week. For transfection, cells were seeded on a 10-cm cell culture dish (Corning, NY) at 2 x 106 cells/dish in 10 mL media overnight. Each transfection of ACE2-Fc variant plasmids was performed using 14 µg/dish DNA with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA). All transfections were performed in triplicate in at least three independent experiments. ACE2-Fc variant proteins were determined by the SDS-PAGE.
7
Determination of Ookinete Conversion Rates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Conversion rates to mature ookinetes were determined as described previously (65 (link)). Briefly, 100 µL of mouse blood with 3 to 10% gametocytemia from the WT line or 1 to 3% gametocytemia from the KO line was collected and immediately transferred to a 10-cm cell culture dish (Corning, cat# 801002) containing ookinete culture medium (RPMI 1640, 25 mM Hepes, 10% FCS, 100 mM xanthurenic acid, pH 8.0) to allow gametogenesis, fertilization, and ookinete development at 22 °C (66 (link)). For ookinete conversion analysis, samples from 12-h culture were collected for thin blood films stained by Giemsa solution (Sigma, cat# GS80). The conversion rate to mature ookinetes was calculated from the counts ratio of mature ookinetes (stage V) to total ookinetes (stages I to V). Three biological replicates of these experiments were performed with identical volumes of infected blood.
8
Isolation and Activation of Mouse Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens were isolated from BALB/c mice or C57BL/6J mice. A 70-μm cell filter (Corning) was placed in a 10-cm cell culture dish (Corning), 6 mL lymphocyte separation solution (BioLegend) was added, and mouse spleens were quickly minted and ground. The lymphocyte separation solution was transferred into 15-mL centrifuge tube (Corning), and 1-mL serum-free DMEM (Gibco) was slowly added into centrifugal tube. Samples were centrifuged at 800 x g for 30 minutes at 4°C. The tunica albugnean layer was collected and washed once by adding 5 mL precooled serum-free DMEM (Gibco), and centrifuged at 250 x g for 10 minutes at 4°C. Finally, the supernatant was discarded. For cell activation, the isolated cells were cultured in 8 mL DMEM (Gibco) containing 10% FBS (Gibco), 50 U/mL IL2 (MCE), and 200 μL mouse CD3/CD28 dynbeads (Invitrogen) for 48 hours.
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!