Prepared MGC-803 and PC3 cells (1×106/mL) were washed twice with cold PBS and then re-suspended gently in 500 μL binding buffer.
Prepared MGC-803 cells (1 × 106/mL) were washed twice with cold PBS and then re-suspended gently in 500 μL binding buffer.
Protein pellets were re-suspended gently in 10 ml of buffer containing 20.0 mmol l−1 Tris HCl (pH 7.5), 20 mmol l−1 NaCl, 10.0 mmol l−1 MgCl2; and dialyzed with Biotec symmetric cellulose ester dialysis membrane (8 10 kDa cut off; Spectrum Labs, Rancho Dominguez, CA, USA) at 4 °C against changes of same buffer for 24 h to remove residual (NH4 2SO4.
Finally, fine-grained suspended particles gently settle and cap the flow deposit.
The Choir Academy houses a public middle school, a charter school run by Harlem's Children's Zone, a technical training school run by the Urban Assembly, a middle school and high school of the Democracy Prep Harlem Charter Schools, and a detention center for suspended students gently called the Alternate Learning Center.
Bacteria were collected in 1 ml PBS/Mg/Ca and re-suspended by gently pipetting and pelleted at 3000 rpm for 3 min. After removal of the supernatant, the bacteria were re-suspended in 500 µl of FITC solution and incubated at room temperature with gentle shaking in the dark for 30 min. After washing (5×) the bacteria were re-suspended in PBS/Mg/Ca with 1% BSA to quench remaining FITC.
After added 1 mL cold PBS, the cells were gently suspended and centrifuged at 1000 rpm for 5 min.
Then, the 6-well microplate was firstly washed by 5%(vol/vol) sterile BSA, and was added into 2 aliquots (0.5 ml each) of prepared Arabidopsis mesophyll protoplasts, then, the protoplasts was gently suspended with 1 ml washing and incubation (WI) solution (4 mM MES pH 5.7, 0.5 M mannitol and 20 mM KCl), and was incubated at 22°C.
Cells (5x103 cells) were gently suspended in 37°C RPMI containing 0.66% agar (DNA grade; Difco Bacto Agar; Becton Dickenson and Company, MD) with and without 200 mM methyl sulfone.
Approximately 2×107 cells were harvested from 0.2 ml of culture by centrifugation at 10,000× g for 10 min, gently suspended in 100 µl PBS containing OspC MAb, and incubated for 1 hour at room temperature.
For fluorescent labeling of unfixed spirochetes, approximately 2×107 cells were harvested from 0.2 ml of culture by centrifugation at 4,000×g for 20 min, gently suspended in 100 µl PBS supplemented with 2 µl of mouse anti-DbpA or -DbpB sera, or FlaB mAb preparation, and incubated for 1 hour at room temperature.
