All the samples (10 mL each) were digested with collagenase type I at the concentration of 1 mg/mL (GIBCO Life Technology, Monza, Italy) dissolved in Hank’s Balanced Salt Solution (HBSS, GIBCO Life Technology, Monza, Italy) with 2% Bovine Serum Albumin (BSA, GIBCO Life Technology, Monza, Italy) for 45 min at 37 °C. Complete culture medium (Dulbecco’s Modified Eagle’s Medium (DMEM), Sigma-Aldrich, Milan, Italy), supplemented with 10% of Fetal Bovine Serum (FBS, GIBCO Life Technologies, Waltham, MA, USA), 1% 1:1 penicillin/streptomycin (P/S solution, GIBCO Life Technologies, Waltham, MA, USA) and 0.6% Amphotericin B (GIBCO Life Technologies, Waltham, MA, USA), was added to neutralize the enzyme action. After the neutralization process, the sample was centrifuged at 3000 rpm for 5 min. The cell pellet was incubated with erythrocyte lysis buffer 1× (Macs Miltenyi Biotec, Milan, Italy) for 10 min at room temperature. Again, the cell suspension was centrifuged and resuspended in a complete culture medium. Finally, the cells were filtered through a 70 µm nylon mesh. The products obtained were then cultured for the following cellular analysis.
Erythrocyte lysis buffer 1
Manufactured by Miltenyi Biotec
Sourced in Italy
Erythrocyte lysis buffer 1× is a laboratory reagent used to selectively lyse red blood cells (erythrocytes) in a sample. It is a balanced salt solution that effectively removes erythrocytes, allowing for the isolation and analysis of other cell types, such as leukocytes, from a heterogeneous cell population.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 1, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Amphotericin b, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Penicillin streptomycin solution p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
2 protocols using erythrocyte lysis buffer 1
1
Isolation and Culture of Adipose-Derived Cells
2
Enzymatic Digestion of Nanofat-Derived Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The remaining portion of lipoaspirate (10 ml) was first processed with the
Hy-Tissue Nanofat kit, followed by the treatment with
collagenase type I. The Nanofat-SVF obtained, as described section “Adipose
Tissue Collection,” was incubated with collagenase type I at a concentration of
1 mg/ml (GIBCO Life Technologies) dissolved in HBSS (GIBCO Life Technologies)
with 2% of BSA (GIBCO Life Technologies) for 45 min at 37°C. Complete culture
medium (DMEM; Sigma-Aldrich), supplemented with 10% of FBS (GIBCO Life
Technologies), 1% of 1:1 penicillin/streptomycin (P/S solution; GIBCO Life
Technologies), and 0.6% of amphotericin B (GIBCO Life Technologies), was added
to neutralize the enzyme action. After the neutralization process, the sample
was centrifuged at 3,000 × g for 5 min. The cell pellet was
incubated with 1 ml of erythrocyte lysis buffer 1× (Macs Miltenyi Biotec) for 10
min at room temperature. Again, the cell suspension was centrifuged and
resuspended with 1 ml of complete culture medium. Finally, the cells were
filtered through a 70-µm nylon mesh.
The product obtained by this method was named “enzymatic digestion of Nanofat-SVF
(N-ED-ASCs).”
Table 1 summarizes
the code names used to identify the different processes employed to treat the
fat tissue.
Hy-Tissue Nanofat kit, followed by the treatment with
collagenase type I. The Nanofat-SVF obtained, as described section “Adipose
Tissue Collection,” was incubated with collagenase type I at a concentration of
1 mg/ml (GIBCO Life Technologies) dissolved in HBSS (GIBCO Life Technologies)
with 2% of BSA (GIBCO Life Technologies) for 45 min at 37°C. Complete culture
medium (DMEM; Sigma-Aldrich), supplemented with 10% of FBS (GIBCO Life
Technologies), 1% of 1:1 penicillin/streptomycin (P/S solution; GIBCO Life
Technologies), and 0.6% of amphotericin B (GIBCO Life Technologies), was added
to neutralize the enzyme action. After the neutralization process, the sample
was centrifuged at 3,000 × g for 5 min. The cell pellet was
incubated with 1 ml of erythrocyte lysis buffer 1× (Macs Miltenyi Biotec) for 10
min at room temperature. Again, the cell suspension was centrifuged and
resuspended with 1 ml of complete culture medium. Finally, the cells were
filtered through a 70-µm nylon mesh.
The product obtained by this method was named “enzymatic digestion of Nanofat-SVF
(N-ED-ASCs).”
the code names used to identify the different processes employed to treat the
fat tissue.
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