Biomedical Engineering Reference
In-Depth Information
10
7
cellsin1-mlculture
medium per 12 ml total gradient volume and 0.5 ml sample (2 -
10 mg protein per milliliter) per 10 ml gradient volume. If self-
generating gradients are used, separation is done with a g
The sample should contain about 8
×
·
t-value
10
6
g
of 1.35
min (30 000 rpm,45min), in case of pre-formed
gradients run with 800 g
av
for 15 min. Allow the rotor to decelerate
without brake to avoid swirling of the bands.
Collect the fraction containing the wanted cells. Dilute this
fraction with a fivefold volume of Soln. B and harvest the cells by
centrifugation at 200
×
·
gfor20min. Wash once with Soln. B.
Free subcellular particles or virus from Percoll by centrifuging
×
Pelleting Percoll
the respective fraction of cell fractionation in a fixed-angle rotor at
100 000
gfor90min. Percoll forms a solid pellet which contains
practically no material of interest.
×
References
Amersham Biosciences (2001) Percoll: Methodology and applications. Edi-
tion AC. Amersham Biosciences AB, Uppsala;
www.amershambiosciences.com
Graham J (2001) Biological centrifugation. BIOS, Oxford, p 85
5.3.5.3 Preparation of Human Lymphocytes
A
3.8% sodium citrate (w/v) in ddH
2
O
Solutions/Reagents
RPMI medium, 10 mM HEPES
6
B
/
C
RPMI medium, 5% FCS (v/v), 0.35 ml
100 ml 2-mercapto-
/
/
ethanol,
10 U
100 ml
streptomycin,
10 U
100 ml
penicillin,
/
29 mg
100 ml l-glutamine
/
D
IMDM medium, 10% human AB serum (v/v), 0.35 ml
100 ml
/
/
2-mercaptoethanol, 10 U
100 ml streptomycin, 10 U
100 ml
/
penicillin, 29 mg
100 ml l-glutamine
E
0.5% Trypan blue (C.I. 23850) in PBS
Important
:Allsolutionshavetobefiltratedsterile.
Mix 10 ml freshly collected venous peripheral blood intensively
with 2.5 ml Soln. A at RT. Then add 12.5 ml Soln. B, warmed to RT.
Carefullycoverthetopof25ml of Lymphoprep (
ρ
=
/
1.077 g
ml)
withthedilutedbloodandspinwith800
×
g (about 1600 rpm)at
20
◦
C for 30 min.
Discard the supernatant and harvest the cells. Dilute the cell
suspension with the same volume of Soln. B and centrifuge at 20
◦
C
with 500
g (1000 rpm)for10min. Dilute the sediment with 30 ml
of Soln. B and spin again.
×
6
RPMI, modified McCoy 5 A medium for cell culture; HEPES, 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid; IMDM, Iscov's mo di-
fied Dulbecco's medium; FCS, fetal calf serum
