Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4–0.7 μl with a sorting speed of 15–20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

1.
V.
Lecault
,
A. K.
White
,
A.
Singhal
, and
C. L.
Hansen
,
Curr. Opin. Chem. Biol.
16
,
381
(
2012
).
2.
A.
Ståhlberg
,
C.
Thomsen
,
D.
Ruff
, and
P.
Åman
,
Am. Assoc. Clin. Chem.
58
,
1682
(
2012
).
3.
A.
Raj
,
C. S.
Peskin
,
D.
Tranchina
,
D. Y.
Vargas
, and
S.
Tyagi
,
PLoS Biol.
4
,
e309
(
2006
).
4.
H. H.
Chang
,
M.
Hemberg
,
M.
Barahona
,
D. E.
Ingber
, and
S.
Huang
,
Nature
453
,
544
(
2008
).
5.
T.
Kalisky
,
P.
Blainey
, and
S. R.
Quake
,
Annu. Rev. Genet.
45
,
431
(
2011
).
6.
K.
Hope
and
M.
Bhatia
,
Nat. Methods
8
,
S36
(
2011
).
7.
N.
Navin
and
J.
Hicks
,
Genome Med.
3
,
31
(
2011
).
8.
D.
Ramsköld
,
S.
Luo
,
Y. C.
Wang
,
R.
Li
,
O.
Deng
,
O.
Faridani
,
G. A.
Daniels
,
I.
Khrebtukova
,
J. F.
Loring
,
L. C.
Laurent
,
G. P.
Schroth
, and
R.
Sandberg
,
Nat Biotechnol.
30
,
777
(
2012
).
9.
A. K.
Shalek
,
R.
Satija
,
X.
Adiconis
,
R. S.
Gertner
,
J. T.
Gaublomme
,
R.
Raychowdhury
,
S.
Schwartz
,
N.
Yosef
,
C.
Malboeuf
,
D.
Lu
,
J. T.
Trombetta
,
D.
Gennert
,
A.
Gnirke
,
A.
Goren
,
N.
Hacohen
,
J. Z.
Levin
,
H.
Park
, and
A.
Regev
,
Nature
498
,
236
(
2013
).
10.
F.
Kamme
,
R.
Salunga
,
J.
Yu
,
D. T.
Tra
,
J.
Zhu
,
L.
Luo
,
A.
Bittner
,
H. Q.
Guo
,
N.
Miller
,
J.
Wan
, and
M.
Erlander
,
J. Neurosci.
23
,
3607
(
2003
).
11.
A.
Ståhlberg
,
D.
Andersson
,
J.
Aurelius
,
M.
Faiz
,
M.
Pekna
,
M.
Kubista
, and
M.
Pekny
,
Nucleic Acids Res.
39
,
e24
(
2011
).
12.
L.
Warren
,
D.
Bryder
,
I. L.
Weissman
, and
S. R.
Quake
,
Proc. Natl. Acad. Sci. U. S. A.
103
,
17807
(
2006
).
13.
S. S.
Rubakhi
and
J. V.
Sweedler
,
Nature Protoc.
2
,
1987
(
2007
).
14.
A.
Bora
,
S. P.
Annangudi
,
L. J.
Mille
,
S. S.
Rubakhin
,
J.
Forbes
,
N. L.
Kelleher
,
M. U.
Gillette
, and
J. V.
Sweedler
,
J. Proteome Res.
7
,
4992
(
2008
).
15.
S. S.
Rubakhi
,
E. V.
Romanova
,
P.
Nemes
, and
J. V.
Sweedler
,
Nat. Methods
8
,
S20
(
2011
).
16.
F.
Tang
,
C.
Barbacioru
,
Y.
Wang
,
E.
Nordman
,
C.
Lee
,
N.
Xu
,
X.
Wang
,
J.
Bodeau
,
B. B.
Tuch
,
A.
Siddiqui
,
K.
Lao
, and
M. A.
Surani
,
Nat. Methods
6
,
377
(
2009
).
17.
K.
Kurimoto
,
Y.
Yabuta
,
Y.
Ohinata
,
Y.
Ono
,
K. D.
Uno
,
R. G.
Yamada
,
H. R.
Ueda
, and
M.
Saitou
,
Nucleic Acids Res.
34
,
e42
(
2006
).
18.
F.
Tang
,
K.
Lao
, and
M. A.
Surani
,
Nat. Methods
8
,
S6
(
2011
).
19.
C. M.
Hempel
,
K.
Sugino
, and
S. B.
Nelson
,
Nature Protocols
2
,
2924
(
2007
).
20.
J. S.
Jang
,
V. A.
Simon
,
R. M.
Feddersen
,
F.
Rakhshan
,
D. A.
Schultz
,
M. A.
Zschunke
,
W. L.
Lingle
,
C. P.
Kolbert
, and
J.
Jen
,
BMC Genomics
12
,
144
(
2011
).
21.
J.
Melin
and
S. R.
Quake
,
Annu. Rev. Biophys. Biomol. Struct.
36
,
213
(
2007
).
22.
M.
Hosokawa
,
A.
Arakaki
,
M.
Takahashi
,
T.
Mori
,
H.
Takeyama
, and
T.
Matsunaga
,
Anal. Chem.
81
,
5308
(
2009
).
23.
A.
Schneider
,
D.
Spitkovsky
,
P.
Riess
,
M.
Molcanyi
,
N.
Kamisetti
,
M.
Maegele
,
J.
Hescheler
, and
U.
Schaefer
,
PLoS ONE
3
,
e3788
(
2008
).
24.
Z.
Környei
,
S.
Beke
,
T.
Mihálffy
,
M.
Jelitai
,
K. J.
Kovács
,
Z.
Szabó
, and
B.
Szabó
,
Sci. Rep.
3
,
1088
(
2013
).
25.
N.
Yoshimoto
,
A.
Kida
,
X.
Jie
,
M.
Kurokawa
,
M.
Iijima
,
T.
Niimi
,
A. D.
Maturana
,
I.
Nikaido
,
H. R.
Ueda
,
K.
Tatematsu
,
K.
Tanizawa
,
A.
Kondo
,
I.
Fujii
, and
S.
Kuroda
,
Sci. Rep.
3
,
1191
(
2013
).
26.
See supplementary material at http://dx.doi.org/10.1063/1.4893922 for a detailed description of experimental methods; table of deposited drop volumes; and a review of possible physical effects in the background of the anomalous pressure we observed.
27.
R. C.
Tolman
,
J. Chem. Phys.
17
,
333
(
1949
).
28.
G.
Ramon
and
A.
Oron
,
J. Colloid Interface Sci.
327
,
145
(
2008
).
29.
J. L.
Pérez-Díaz
,
M. A.
Álvarez-Valenzuela
, and
J. C.
García-Prada
,
J. Colloid Interface Sci.
381
,
180
(
2012
).
30.
N.
Orgovan
,
B.
Peter
,
S.
Bősze
,
J. J.
Ramsden
,
B.
Szabó
, and
R.
Horvath
,
Sci. Rep.
4
,
4034
(
2014
).
31.
S.
Faraasen
,
J.
Vörös
,
G.
Csúcs
,
M.
Textor
,
H. P.
Merkle
, and
E.
Walter
,
Pharm. Res.
20
,
237
(
2003
).
32.
R.
Salánki
,
C.
Hős
,
N.
Orgovan
,
B.
Péter
,
N.
Sándor
,
Z.
Bajtay
,
A.
Erdei
,
R.
Horvath
, and
B.
Szabó
, “
Single cell adhesion assay using computer controlled micropipette
,”
PLoS ONE
(to be published).

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