Lung tissue was excised from an anesthetized mouse (1-week-old), minced, and then incubated in Dulbecco’s Modified Eagle’s Medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 1 mg/ml collagenase/dispase solution (Roche, Basel, Switzerland) and 5 U/ml DNase I (Roche) for 45 min at 37 °C. The digested pieces were further minced by passing them through a 20-gauge needle and then filtered with a 70-μm cell strainer (BD Biosciences, San Jose, CA, USA). The filtrate was centrifuged for 5 min at 400×g at 20 °C, and the resulting cell pellet was suspended in PBS containing 0.1% BSA. The cells were incubated with Dynabeads (Invitrogen, Carlsbad, CA, USA) precoated with rat anti-mouse CD31 antibody (BD Biosciences) for 30 min at room temperature. ECs bound to the Dynabeads were collected with a magnet, washed using PBS with 0.1% BSA, and then cultured in a 60-mm dish coated with 2% gelatin in Endothelial Cell Growth Medium 2 (Takara). The purity of isolated ECs was > 95%, which was confirmed by immunofluorescence microscopy using anti-CD31 and anti-CD102 antibodies (BD Biosciences).
Dynabeads
Manufactured by Takara Bio
Sourced in Japan
Dynabeads are a versatile line of magnetic beads used in a variety of applications for the isolation, purification, and manipulation of biological molecules and cells. The core function of Dynabeads is to provide a solid-phase support that can be easily separated from a liquid sample using a magnetic field.
Automatically generated - may contain errors
Lab products found in correlation
Dynabead, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd31 antibody, by BD (1 mentions)
Dnase 1, by Roche (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Anti cd31 and anti cd102 antibodies, by BD (1 mentions)
Collagenase dispase solution, by Roche (1 mentions)
Endothelial cell growth medium 2, by Takara Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Retronectin, by Takara Bio (1 mentions)
2 protocols using dynabeads
1
Isolation of Mouse Lung Endothelial Cells
2
Isolation and Transduction of Primary Human T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral
blood mononuclear cells (PBMCs) were isolated by density gradient
centrifugation from apheresis products of healthy donors (Brigham
and Women’s Hospital Crimson Core, Boston, MA). Primary human
T cells were purified from PBMCs by Pan T Cell Isolation Kit (Miltenyi
Biotec, Germany). Purified T cells were stimulated with anti-CD3/CD28
Dynabeads (Thermo Fisher Scientific) at a T cell/bead ratio of 1:2.
After 24 h, Dynabeads were removed and stimulated T cells were seeded
on a Retronectin (Takara Bio, Japan)-coated nontissue culture-treated
plate with virus and centrifuged at 1200g, 32 °C
for 30 min. One-time infection was carried out for the smaller vectors
shown inFigures 1 and 2 at a multiplicity of infection (MOI) of 5–10.
Larger vectors shown inFigures 3 and 4 were infected at a MOI
of 10–20, spread out over 2 days of daily infection. During
stimulation and infection, T cells were supplemented daily with 100
U/mL of IL-2 and 10 ng/mL of IL-15 (NCI Preclinical Repository, Frederick,
MD). After infection, T cells were maintained by supplementing with
100 U/mL of IL-2 and 10 ng/mL of IL-15 every 3 days. T cells were
expanded for another 5–6 days after the last infection prior
to use in experiments.
blood mononuclear cells (PBMCs) were isolated by density gradient
centrifugation from apheresis products of healthy donors (Brigham
and Women’s Hospital Crimson Core, Boston, MA). Primary human
T cells were purified from PBMCs by Pan T Cell Isolation Kit (Miltenyi
Biotec, Germany). Purified T cells were stimulated with anti-CD3/CD28
Dynabeads (Thermo Fisher Scientific) at a T cell/bead ratio of 1:2.
After 24 h, Dynabeads were removed and stimulated T cells were seeded
on a Retronectin (Takara Bio, Japan)-coated nontissue culture-treated
plate with virus and centrifuged at 1200g, 32 °C
for 30 min. One-time infection was carried out for the smaller vectors
shown in
Larger vectors shown in
of 10–20, spread out over 2 days of daily infection. During
stimulation and infection, T cells were supplemented daily with 100
U/mL of IL-2 and 10 ng/mL of IL-15 (NCI Preclinical Repository, Frederick,
MD). After infection, T cells were maintained by supplementing with
100 U/mL of IL-2 and 10 ng/mL of IL-15 every 3 days. T cells were
expanded for another 5–6 days after the last infection prior
to use in experiments.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!