Air-liquid interface (ALI) culture of epithelial clones was performed as described (Kumar et al., 2011 (link); Zuo et al., 2015 (link)). Briefly, Transwell inserts (Corning, USA) were coated with 20% Matrigel (BD biosciences, USA) and incubated at 37 °C for 30 min to polymerize. 200,000 irradiated 3T3-J2 cells were seeded to each Transwell insert and incubated at 37 °C, 7.5 % CO2 incubator overnight. QuadroMACS Starting Kit (LS) (Miltenyi Biotec, Germany) was used to purify epithelial by removal of feeder cells. 200,000–300,000 cells were seeded into each Transwell insert and cultured in StemECHO™PU media. At confluency (3–7 days), the apical media on the inserts was removed through careful pipetting and the cultures were continued in differentiation media (PneumaCult-ALI Media, STEMCELL Technologies, Vancouver) for an additional 14–21 days prior to harvesting. The differentiation media was changed every one or two days.
Quadromacs starting kit ls
Manufactured by Miltenyi Biotec
Sourced in Germany
The QuadroMACS Starting Kit (LS) is a laboratory equipment product from Miltenyi Biotec. It is designed to facilitate cell isolation and purification processes. The core function of this kit is to provide the necessary components and tools to perform magnetic-activated cell sorting (MACS) separations.
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Lab products found in correlation
4 protocols using quadromacs starting kit ls
1
Air-Liquid Interface Culture of Epithelial Cells
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Intestinal Epithelial Cell Air-Liquid Interface
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Air-liquid interface (ALI) culture of intestinal and colonic epithelial cells was performed as described12 (link),51 (link). Briefly, Transwell inserts (Corning, USA) were coated with 20% Matrigel (BD Biosciences, USA) and incubated at 37 °C for 30 min to polymerize. 200,000 irradiated 3T3-J2 cells were seeded to each transwell insert and incubated at 37 °C, 7.5% CO2 incubator overnight. QuadroMACS Starting Kit (LS) (Miltenyi Biotec, Germany) was used to purify the stem cells by removal of feeder cells. 200,000–300,000 stem cells were seeded into each Transwell insert and cultured with SCM-6F8. At confluency (3–7 days), the apical media was removed through careful pipetting and the cultures were continued for an additional 6–12 days before analysis.
3
Isolation of CD14+ Monocytes from PBMCs
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All blood samples were collected in EDTA pre-coated tubes from healthy volunteers under an IRB-approved protocol (STUDY0004777) and processed immediately after collection. PBMCs were isolated from whole blood by density separation over a solution of 1-Step™ Polymorph (Accurate Chemical & Scientific Co., AN221725) at 500×g for 30 min without brakes. The white buffy PBMC layer was then washed twice in Wash buffer (HBSS without Ca2+ and Mg2+ with 10mM HEPES and 5mg/ml BSA) and centrifuged at 350×g for 7 min without brakes to remove platelets. Red blood cells were lysed by exposing the washed PBMC layer to 1/6x PBS for 1 min, followed by 4x PBS for 1 min, at a 3:1 ratio, and spinning at 350×g for 7 min. PBMCs were resuspended in Wash buffer and spun down at 350×g for 7 min to remove any remaining PBS. PBMCs were resuspended in an Isolation buffer (1XDPBS without Ca2+ and Mg2+ with 2mM EDTA and 1 mg/ml BSA). CD14+ Monocytes were isolated from PBMCs by positive magnetic enrichment using the QuadroMACS Starting Kit (LS) (Miltenyi Cat# 130–091-051) using the manufacturer’s protocol.
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Intestinal Epithelial Cell Air-Liquid Interface
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