Human monocytes were isolated from human buffy coats purchased from the Massachusetts General Hospital blood bank using a standard Ficoll gradient and subsequent CD14+ cell positive selection (Stemcell Technologies). Selected monocytes were cultured in ultra low- adherence flasks (Corning) for 0, 3, or 6 days with RPMI media (Invitrogen) supplemented with 10% FBS (Invitrogen) and 50 ng/mL human M-CSF (Biolegend). SeqWell analysis was performed as previously described54 . Briefly, at the respective timepoint, cells were detached using trypsin, spun down, and counted. Approximately 12,000 cells were loaded on each array for each timepoint and condition to minimize doublet-loading. The arrays were sealed with a semi-permeable membrane prior to cell lysis and hybridization to single-cell beads. Beads were subsequently pooled for reverse transcription and whole transcriptome amplification.
Ultra low adherence flasks
Manufactured by Corning
Ultra low-adherence flasks are specialized laboratory equipment designed to prevent cell attachment to the vessel surface. They feature a unique surface treatment that minimizes cell adherence, promoting the growth and maintenance of cells in suspension culture.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi media, by Thermo Fisher Scientific (2 mentions)
Cd14 cell positive selection, by STEMCELL (2 mentions)
Human m csf, by BioLegend (1 mentions)
Ficoll gradient, by STEMCELL (1 mentions)
Human gm csf, by BioLegend (1 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (1 mentions)
3 protocols using ultra low adherence flasks
1
Isolation and Profiling of Human Monocytes
2
Monocyte Isolation and Differentiation
3
THP-1 and hCMEC/D3 Cell Culture
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The human monocytic cell line THP-1 (TIB-202, from the ATCC) was cultured and differentiated as described in reference 61 (link). These cells were negative for mycoplasma. For the model BBB, we obtained the immortalized human brain endothelial cell line hCMEC/D3, described and reviewed in reference 22. These cells were obtained from Babette Weksler (Cornell University Medical College) and were grown as described in reference 23. Primary blood monocytes were isolated by negative immunoselection (Pan Monocyte isolation kit; Miltenyi Biotec, Inc.) from peripheral blood mononuclear cells (PBMCs) that were obtained from leukoreduction (LRS) chambers following routine platelet donations at the apheresis center at Washington University School of Medicine. The cells were kept in suspension by growth in ultra-low-adherence flasks (Corning) in human peripheral blood monocyte (hPBM) medium (RPMI medium with l -glutamine and sodium bicarbonate, 10% fetal bovine serum [FBS], 1 mM sodium pyruvate, 1× penicillin-streptomycin [PenStrep], 1% nonessential amino acids [NEAA], and 10 mM HEPES [pH 7]).
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