HeLa cells grown in 150-mm dishes (Corning, United Kingdom) were infected with S. Typhimurium for 14 h. Phosphate-buffered saline (PBS)-washed cells were scraped with a rubber policeman, centrifuged for 5 min at 100 × g, and resuspended in 500 µl of lysis buffer (PBS–5% glycerol–0.5% Triton X-100–1 mM phenylmethylsulfonyl fluoride [PMSF]) for 30 min at 4°C. Lysates were centrifuged for 10 min at 16,000 × g to remove debris before being precleaned with 20 µl of protein G agarose for 1 h (Pierce). The precleaned lysate was mixed with 40 µl of anti-HA antibody-conjugated agarose (Pierce) or anti-Flag antibody-conjugated M2 agarose (Sigma) and incubated at 4°C for 2 h to immunoprecipitate HA-tagged SseF or Flag-tagged SseG. The immunoprecipitates were washed 4 times with lysis buffer and then eluted with 50 µl of 2 mg/ml HA peptide or 0.1 mg/ml Flag peptide. Sample proteins were separated by SDS-PAGE and analyzed by immunoblotting with appropriate antibodies.
150 mm dishes
Manufactured by Corning
Sourced in United States
The 150-mm dishes are laboratory equipment used for various applications in scientific research and experimentation. They provide a standardized container for cell culture, sample preparation, and other experimental procedures. The dishes are made of durable materials and have a consistent diameter of 150 millimeters, allowing for controlled and reproducible experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2 g, by Addgene (2 mentions)
Anti flag antibody conjugated m2 agarose, by Merck Group (1 mentions)
Cellcarrier ultra plates, by PerkinElmer (1 mentions)
Polyvinylidene fluoride filter, by Merck Group (1 mentions)
Lentivirus precipitation solution, by Merck Group (1 mentions)
Opti mem reduced serum media, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Sh800s cell sorter, by Sony (1 mentions)
Viral production medium, by Lonza (1 mentions)
Durapore pvdf membrane, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
5 protocols using 150 mm dishes
1
Immunoprecipitation of Salmonella Effectors
2
Transfection of HEK293S cells with CGRP receptor
3
Lentiviral Transduction of HEK293T and A549 Cells
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On day 1, one confluent 100mm dish (Corning) of HEK293T cells were seeded into three 150mm dishes (Corning). On day 2, cells were ∼50%–70% confluent at the time of transfection. For each dish, 27.18 μg of pHR vector containing the construct of interest, 23.76 μg of dR8.91 and 2.97 μg of pMD2.G (Addgene) were mixed in 4.5 mL of Opti-MEM reduced serum media (GIBCO) with 150 μL of Mirus TransIT-LT1 reagent and incubated at room temperature for 30 minutes. The transfection complex solution was distributed evenly to HEK293T cultures dropwise. On day 5, lentiviruses are harvested from the supernatant with a sterile syringe and filtered through a 0.22-μm polyvinylidene fluoride filter (Millipore).
For A549 cells, lentivirus precipitation solution (Alstem) was added and precipitated as per the manufacturer’s protocol. One well of a 6-well plate (Corning) of A549 cells at ∼50% confluency were transduced with the precipitated lentivirus. After 2-3 days of growth, the cell supernatant containing the virus was removed and the cells were expanded. Cells were then sorted for mCherry+ cells using a Sony SH800S cell sorter.
For A549 cells, lentivirus precipitation solution (Alstem) was added and precipitated as per the manufacturer’s protocol. One well of a 6-well plate (Corning) of A549 cells at ∼50% confluency were transduced with the precipitated lentivirus. After 2-3 days of growth, the cell supernatant containing the virus was removed and the cells were expanded. Cells were then sorted for mCherry+ cells using a Sony SH800S cell sorter.
4
Lentiviral Vector Production in HEK293T Cells
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HEK293T cells were grown on 150-mm dishes (CORNING) coated with poly-l -lysine (Sigma) until 90% confluency. For each 150-mm dish, 21 μg of the library plasmids, and 15 μg of psPAX2 (Addgene 12,260) and 6 μg of pMD2.G (Addgene 12,259) were transfected into HEK293T cells using 63 μl of Neofect DNA transfection reagent (Neo Biotech). At 16 h post transfection, the culture medium was changed with viral production medium (Lonza). Virus supernatant was collected 40 h post transfection, filtered with a 0.45-μm Sterile Filter Unit with Durapore PVDF Membrane (Millipore), aliquoted, and stored at − 80 °C before use.
5
MLO-Y4 Cell Conditioning with AD
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