CelLytic™ MT Cell Lysis Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- CD14+ cells were isolated directly from whole blood with Dynabeads® CD14.	- For extraction follow the manufacturer's protocol.

Upstream tips
- CD14+ cells were isolated directly from whole blood with Dynabeads® CD14.
Protocol tips
- For extraction follow the manufacturer's protocol.

Publication protocol

Blood samples were processed immediately after collection at baseline and altitude study sites. CD14+ cells were isolated directly from whole blood with Dynabeads® CD14 (Invitrogen Dynal AS, Oslo, Norway); 125 µl of Dynabeads (4×108/ml) was used per 1.5 ml blood. The Dynabeads and the whole blood were incubated in 1.5 ml tubes for 20 minutes with gentle rotation and afterwards the tubes were placed in a magnet for 2 minutes (MPC 6; Dynal). For positive isolation of PBM, the supernatants were discarded and the beads with attached CD14+ cells were washed three times by resuspending in phosphate buffer saline, and the beads with attached cells were frozen. During the transport from the study sites at high altitude to the laboratory in Switzerland, the blood samples were kept at −30°C. During the rapid descent to the basecamp, the samples were kept in a pre-cooled insulation container. For helicopter transport from the basecamp to the nearest hospital (Bishkek), the samples were placed in a refrigerator. A commercial refrigerated transport service with standard temperature monitoring was used to transfer the samples from Kyrgyzstan to the laboratory in Switzerland. After being transported to the laboratory, the beads were lysed by the addition of 100 μl of CelLytic™ MT cell lysis reagent (Sigma-Aldrich, St Louis, MO, USA, C3228). The protein concentration of monocyte lysates was determined with the Quant-iTM assay kit and read with the Qubit-TM fluorometer (Invitrogen, Basel, Switzerland). The lysates were frozen at −80°C for enzymatic activity measurements.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for CelLytic™ MT Cell Lysis Reagent below.

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