Table 2 Procedural steps for isolation of primary astrocytes, pericytes, and endothelial cells highlights variations in details of conserved steps between many previously published primary BBB cell isolation protocols and includes references where each of the protocol details are featured

 Astrocytes

Mouse: 0 days

[36]

Remove surface vessels

[1, 3, 29, 37], [36],

Mouse: 1–4 days

[3, 37]

Mince + pipette trituration

[1, 29,30,31], [38],

Rat (SD): 0–2 days

[31]

Enzyme incubation: trypsin, 30 min, 37 °C

[3, 31],

Rat (W): 1–3 days

[1]

Enzyme incubation: DNAse + trypsin, 20 min, 25 °C

[36]

Rat (SD): 2 weeks

[29]

Enzyme incubation: Collagenase Type II + DNase, 10 min, 25 °C

[37]

Pig: 6 months

[5, 30]

Enzyme incubation: Papain + DNase

[39]

40-70-µm Cell strainer

[1, 29, 31, 39],

Vigorous orbital shaker after 8–14 days

[1, 3, 29, 31],

Tapping or orbital shaker at confluency

[4, 36, 38]

Pericytes & endothelial cells

Mouse: 6–10 weeks

[37, 40,41,42,43,44,45]

Euthanasia method (CO₂ or isofluorane)

[2, 29, 31, 37, 40, 45]

Mouse: 40–52 weeks

[40]

Roll brain on sterile gauze, blotter paper, filter paper, or cellulose chromatography paper to remove leptomeningeal cells

[4, 31, 37, 40, 41],

Rat (W): 3 weeks

[1]

Manually remove meninges with forceps

[1, 29,30,31, 37, 38]

Rat (W): 8 weeks

[1]

Mince + pipette trituration (1mm³ pieces)

[4, 29, 31, 37, 38, 40, 41, 45]

Rat (SD): 2–3 weeks

[29, 30]

Mince with tissue grinder

[40]

Rat (SD): 3 months

[20]

1st Enzymatic Digestion: DNAse 1 + TLCK + collagenase/dispase 1 h, 37 °C

[31, 40]

1st Enzymatic Digestion: MEM + papain and DNase I, 1 h 10 min, 37 °C

[43, 45]

1st Enzymatic Digestion: Collagenase Type II (1 mg/mL) 1.5 h, 37 °C

[1]

1st Enzymatic Digestion: Collagenase Type II (1 mg/mL) +

DNAse (15 µg/mL) 1–1.5 h, 37 °C

[4, 30, 38]

1st Enzymatic Digestion: Collagenase Type II (1 mg/mL) + DNAse 1.25 h, 37 °C, on benchtop orbital shaker

[37, 41]

1st Enzymatic Digestion: Collagenase/Dispase (270 U/mL and 0.1%) + DNAse (10 U/mL) 1.5 h, 37 °C

[29, 42, 43]

18% Dextran in DMEM, vortex, add enzymes, then centrifuge 6080 x g, 10 min, 4 °C.

[40]

20–30% BSA in DMEM, then centrifuge ~ 1000xg, 15–20 min

[1, 4, 29,30,31, 37, 38, 41, 43, 45]

2nd 20–30% BSA separation

[31, 37]

2nd Enzymatic Digestion: Collagenase/Dispase (1 mg/mL), 1 h, 37 °C,

[1, 4, 29]

2nd Enzymatic Digestion: Collagenase/Dispase (1 mg/mL) + DNAse (6.7 µg/ml in DMEM 50 min–1 h, 37 °C

[30, 37, 38, 41]

33% Continuous Percoll separation

[1, 4, 30, 31, 37, 38, 41,42,43]