Magnetic Cell Isolation via Insulin Staining

As previously described [12 (link), 26 (link)], cells were stained at 4°C with 0.1 μg insulin– biotin per 10⁶ cells per 100 μl and antibodies (as described below) for 30 min, fixed with 2% (wt/vol.) formaldehyde and incubated with streptavidin–Alexa⁶⁴⁷ (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min. Cells were then washed and incubated with anti-Cy5/anti-Alexa⁶⁴⁷ microbeads (Miltenyi) for 10 min and passed over magnetised LS columns (Miltenyi). Columns were washed three times, and bound cells were eluted in 6 ml of MACS buffer.

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Smith M.J., Hinman R.M., Getahun A., Kim S., Packard T.A, & Cambier J.C. (2018). Silencing of high-affinity insulin-reactive B lymphocytes by anergy and impact of the NOD genetic background in mice. Diabetologia, 61(12), 2621-2632.

Publication 2018

streptavidin–Alexa647 anti-Cy5/anti-Alexa647 microbeads

Alexa647 Antibodies Biotin Buffer Cells Formaldehyde Insulin cells Microbeads Streptavidin

Corresponding Organization :

Other organizations : University of Colorado Denver

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Variable analysis

independent variables

Insulin-biotin staining concentration (0.1 μg per 10^6 cells per 100 μl)
Incubation time with insulin-biotin (30 min)
Incubation time with streptavidin-Alexa647 (15 min)
Incubation time with anti-Cy5/anti-Alexa647 microbeads (10 min)

dependent variables

Proportion of cells bound to magnetised LS columns

control variables

Temperature (4°C)
Formaldehyde concentration (2% wt/vol.)
MACS buffer used for washing and elution

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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