Stimulation of basophils + a bunch of other cells in vitro

https://ars.els-cdn.com/content/image/1-s2.0-S2589004220309214-mmc1.pdf

One milliliter aliquots of whole blood were distributed into round-bottom tubes with loose lids and warmed in a 37°C water bath for 30 seconds. Freshly prepared, 1 mL aliquots of pre-warmed RPMI 1640 (Gibco) were supplemented with polyclonal rabbit anti-IgE (Bethyl Laboratories) or IL-3 (PeproTech). The prewarmed 1 mL of whole blood was then mixed with either 1 mL of RPMI 1640, or RPMI 1640 supplemented with anti-IgE (final concentration, 1 μg/mL) or IL-3 (final concentration, 2 ng/mL) and incubated for 30 minutes at 37°C in a 5% CO2 incubator. After the incubation period, red blood cells were removed by hypotonic lysis with 20 mL of cold RBC lysis buffer (BioLegend) and incubation for 15 minutes on ice. The lysis reaction was stopped with PBS supplemented with EDTA (Invitrogen Life Technologies; final concentration, 2 mM) followed by centrifugation at 250g for 5 minutes at 4°C (all centrifuge runs on live cells were done with these conditions).

*Blood was stored at most 24h at 4 deg C

*Anticoagulant EDTA or heparin can have distinct impact on basophil activation profiles

*We chose to use whole blood vs. cells post red blood cell lysis because we wanted to retain as close of an environment as that in situ

*Must use sterile techniques

*If followed by mass cytometry analysis vs flow cytometry, watch out for metal contamination. Must use low barium PBS, avoid glass bottles

Article Titlehttps://ars.els-cdn.com/content/image/1-s2.0-S2589004220309214-mmc1.pdf

Abstract

We used stimulation with anti-IgE or IL-3 in whole blood, which would allow for the potential interaction of multiple cell types under conditions that simulate an allergic response.


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