Synaptotagmin-1 and -7 are functionally overlapping Ca 2+ sensors for exocytosis in adrenal chromaffin cells

Schonn et al. 10.1073/pnas.0712373105.

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SI Figure 6
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SI Figure 6

Fig. 6. The exocytotic phenotype of Syt7 -/- mice is found also in embryonic cells. (A and B) Embryonic cells from WT (Syt 7 +/+, black; n = 19, N = 2) and Syt 7 KO littermates (Syt 7 -/-, gray trace, n = 29, N = 2) were recorded for control purpose. Similar phenotype as in adult animals was found, i.e., a reduction in burst and sustained components amplitude (compare Fig. 3) and a speed-up of the remaining burst component.





SI Materials and Methods

For adult mice (Syt 7 KO and Syt 7 KI strains), adrenal glands were dissected and cleaned in filtered Locke's solution [154 mM NaCl, 5.6 mM KCl, 0.85 Mm NaH2PO4, 10 mM glucose (pH 7.0)]. The glands were gently opened with tweezers before digestion (40 min, 37°C) in 1 ml of papain solution (20-25 units/ml in DMEM supplemented with 200 mg/liter L-cystein, 1 mMCaCl2, 20 mM EDTA, equilibrated in 5%CO2/95% O2; the papain was from Worthington). The papain activity was inactivated for 5 min by addition of 750 ml of DMEM supplemented with 10% heat-inactivated FCS (Invitrogen), 2.5 g/liter albumin, 2.5 g/liter trypsin inhibitor (Sigma). For embryonic glands, the volumes of papain and inactivating solutions were reduced to 200 and 175 ml, respectively, and the glands were left intact. The solution was carefully removed and replaced by 600 ml (adults) or 200 ml (embryos) of complete culture medium (DMEM, containing 0.4% penicillin/streptomycin and 1% ITSX; all from Invitrogen).

Digested glands were gently triturated by passing them several times through a 1-ml pipette-tip opening (for adults; 200-ml tip for embryonic glands). In the case of embryonic glands, the cell suspension was directly dispensed on uncoated glass coverslips (50 ml each). The cell suspension obtained from adult glands was briefly centrifuged (4 min, 180 ´ g), the supernatant carefully removed, and the pellet gently dispersed in 400-600 ml of complete culture medium. Cell suspension was plated on glass coverslips (100 ml per coverslip). After 30 min, the wells were filled with 2.5 ml of complete culture medium. The cells were incubated at 8% CO2, 37°C, 95% relative humidity and used within 1-4 days.

This Article

  1. Published online before print February 28, 2008, doi: 10.1073/pnas.0712373105 PNAS March 11, 2008 vol. 105 no. 10 3998-4003
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