Two methods were applied. One method was based on the live cell assay, while the other method on the fixed cell assay. For the first method, H441 cells (106/mL) were suspended in RPMI-1640 medium supplemented with 0.1% BSA, distributed (105 cells/100 μL) into 5-mL round bottom polypropylene tubes (Falcon), mixed with 10 μg of PE-conjugated isotype control or anti-human ADAM10 mAb (R&D Systems), incubated for 30 min on ice and washed twice with the same medium used for preparing cell suspension. After washing, cells were either suspended in the medium alone or 10 μM PEPDAB005 solutions, incubated for 30 min at 21oC, and fixed with 2% PFA. For the second method, K562 cells (106/mL) were suspended in Hank's buffer, distributed (105 cells/100 μL) into 5-mL round bottom polypropylene tubes (Falcon), mixed with 10 μg of PE-conjugated IgG control antibody, anti-human ADAM10 antibody and/or 5 μM PEPDAB005 solution, incubated at 0oC for 60 min, washed three times with Hank's buffer, fixed with 0.005% GAL at 0oC for 30 min, incubated at 21oC for 30 min and post-fixed with 2% PFA.
Round bottom polypropylene tubes
Manufactured by BD
Sourced in United States
Round-bottom polypropylene tubes are laboratory equipment used for various applications. They are made of polypropylene, a commonly used plastic material in the laboratory setting. The round-bottom design provides stability and facilitates mixing or centrifugation processes. These tubes are suitable for a range of laboratory procedures that require a durable, chemically resistant, and versatile container.
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Lab products found in correlation
7 protocols using round bottom polypropylene tubes
1
Quantifying ADAM10 Expression in Live and Fixed Cells
2
Isolation of Highly Purified Platelets
3
Isolated Platelets from Healthy Subjects
4
Brucella abortus Infection in THP-1 Cells
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THP-1 cells at a concentration of 0.5 × 106/ml were infected in round-bottom polypropylene tubes (Falcon) with a multiplicity of infection (MOI) of 100 of B. abortus S2308 or GFP-S2308, in presence or absence of platelets (THP-1:PLT ratio 1:100). All infections were performed for 2 or 4 h in standard medium containing no antibiotics. In all cases, cells were then extensively washed to remove uninternalized bacteria, and infected cells were maintained in culture in medium supplemented with 100 µg of gentamicin/ml and 50 µg of streptomycin/ml. At different times post-infection, supernatants were collected, filtered, and stored at −70°C for later determination by Enzyme Linked ImmunoSorbent Assay (ELISA). In another set of experiments, the cells were equally infected with B. abortus in presence or absence of platelets, and the expression of surface markers was evaluated by flow cytometry.
5
Internalization of Brucella abortus in THP-1 cells
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5 × 105.ml−1 THP-1 were infected with a multiplicity of infection (MOI) of 100 B. abortus S2308 per cell in round-bottom polypropylene tubes (Falcon). This procedure was performed with 150 U.ml−1 IFN-γ for 2 h in standard medium without antibiotics. Afterwards, cells were extensively washed in order to remove uninternalized bacteria. Infected cells were maintained in culture in presence of IFN-γ, 100 μg.ml−1 gentamicin, and 50 μg.ml−1 streptomycin for other 48 h.
6
Whole Blood Stimulation Assay
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One milliliter of heparinized blood was added to 12 × 75 mm round-bottom polypropylene tubes (Falcon) as described as previously (72 (link)) and then 1 ml of RPMI with or without 10 ng/ml LPS was added for stimulation in the presence of different concentrations of anti-IL-1R7 or IL-18BP or IL-1Ra. The antibody or IL-18BP or IL-1Ra was added 30 min before the stimuli. The tubes were closed tightly with the caps and were mixed by inversion. Blood was incubated upright in the sealed tubes at 37 °C for 3 days. After incubation at 37 °C, the tubes were inverted several times to mix the formed elements, and Triton X-100 was added (5%; Bio-Rad Laboratories) to a final concentration of 1%. The tubes were again inverted several times until the blood was clarified. The lysed blood was frozen at –70 °C until assay.
7
In Vitro Infection of Immune Cells with Brucella Strains
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THP-1 cells at a concentration of 0.5 x 106/ml were infected in round-bottom polypropylene tubes (Falcon) with different multiplicities of infection (MOI) of B. abortus S2308, B. abortus RB51, B. abortus virB10, B. abortus btpA, B. abortus btpB, B. abortus btpAbtpB or B. abortus Bpe159 mutants. All infections were done in the presence of 150 U/ml IFN-γ (Endogen) for 2 h in standard medium containing no antibiotics. In another set of experiments, BMM from WT or TLR7 KO mice at a concentration of 0.5 x 106/ml were infected in a 24-well plate with different MOI of B. abortus S2308. Infections were done in the presence of 10 ng/ml mIFN-γ (Peprotech) for 2 h in standard medium containing no antibiotics. In all cases, cells were extensively washed to remove uninternalized bacteria and infected cells were maintained in culture in the presence of IFN-γ or mIFN-γ, 100 μg/ml gentamicin and 50 μg/ml streptomycin for an additional 48 h.
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