细胞结合实验
1| Prepare cells for the experiment. This procedure will present the detailed cell-SELEX protocol for both suspension and
adherent cultured cell lines. For adherent cells, there are two options: the experiment can be performed either directly in a
culture dish or using dissociated cells. If using dissociated cells, prepare them by following the steps given in Box 1 before
following the procedure given below. If performing the experiment in a culture dish, refer to Box 2 and continue with the
procedure from Step 61.
Initial Dna library pool preparation ● tIMInG 20 min
准备实验细胞。这个程序将详细的细胞SELEX协议和悬浮
贴壁培养细胞系。贴壁细胞，有两种选择：实验可以直接在
或使用解离的细胞培养皿。如果使用解离的细胞，他们准备通过下面1盒之前的步骤
下面给出的程序如下。如果在培养皿中进行实验，涉及2盒并继续
步骤61步骤。最初DNA文库池制备●定时20分钟
2|  Add 20 µl of 0.5 mM (10 nmol) DNA library to 350 µl of binding buffer, mix and heat the mixture at 95 °C for 5 min.
Snap-cool on ice and keep on ice until ready to use (same day).
pause poInt If selection is not accomplished on the same day, store the DNA library pool at  −  20 °C. Thaw library on ice
whenever ready to use. The denaturation step is not necessary once DNA has been thawed on ice.
crItIcal step Heating the DNA at 95 °C and subsequent fast cooling on ice are important to create folded ssDNA.
preparation of target cells: cell viability ● tIMInG 30 min
添加20µL 0.5毫米（10 nmol）350µL的结合缓冲液DNA文库，混合和热的混合物在95°C 5分钟。
卡酷冰保持在冰上待用（同一天）。
暂停点如果选择不在同一天完成，在−20°C.解冻库在冰店DNA文库池
当准备使用。变性步骤是不必要的一旦DNA已解冻的冰。
关键步骤中加热到95°C和随后的快速冷却的冰上的DNA是创造折叠ssDNA重要。
靶细胞的制备：细胞活力●定时30分钟
3|  Determine cell viability using Trypan blue exclusion assay.
crItIcal step Cell viability assessment is very important, especially for suspension
cells. Too many dead cells will seri-
ously affect the efficiency of selection. DNA can nonspecifically adhere to and enter dead cells. This will cause the loss of
important sequences, delay of enrichment or even the failure of selection. As the highest cell viability is ideal, it is preferable
to use more than 95% viable cells.
preparation of target cells: cell number ● tIMInG 20 min
4|  Determine the concentration of cells using a hemocytometer. On the basis of cell count, determine what volume will
correspond to the number of cells needed for a specifc round of selection.
crItIcal step For the first round of selection, use the highest number of cells possible for collecting specific sequences,
preferably between 5 and 10 million cells.
preparation of target cells: washing by centrifugation ● tIMInG 30 min
5|  Take the cell volume that yields the desired number of cells into a 15-ml centrifuge tube and centrifuge cells at 150g
for 3 min at 4 °C. Remove the supernatant and add 3 ml of washing buffer. Resuspend cells by pipetting up and down,
tapping the bottom of the centrifuge tube or mild vortexing. Pellet the cells at the same speed and duration. Repeat
washing once more.
crItIcal step Avoid strong vortexing as it can cause cell breakage and may eventually affect selection.
Incubation of cells with Dna library pool ● tIMInG 1 h
6|  Resuspend cells in 330 µl of binding buffer. Add all of the 370 µl snap-cooled DNA library (Step 2) to the 330 µl cell
suspension, mix thoroughly and incubate the mixture on ice for 1 h on a rotary shaker. Incubation temperature depends on
BoX 1 | DISSoCIATE ADHERENT CELLS ● tIMInG 4 H 15 mIN
1. Split cells 24 h before dissociation treatment.
2. Dissociate cells from culture fask or dish with nonenzymatic dissociation solution or by short-term (30 s–1 min) trypsin treatment.
After incubation, remove trypsin or dissociation buffer and immediately add cold culture medium containing FBS. Cells are split 24 h
before dissociation; hence, they can be removed by short-term treatment. If cells are left for 2–3 d, this will not be possible.
3. Transfer cells from fask or dish to centrifuge tube for subsequent use. FBS will halt further action of residual trypsin.
4. For cells dissociated with nonenzymatic dissociation buffer, count cells and use required amount for aptamer selection by following
the same procedure as described for suspension cells (Steps 2–60).
5. For cells dissociated by short-term trypsin treatment, use cells immediately, as described (Steps 2–60), or incubate cells under cell
culture conditions, but with rocking for at least 2 h.
crItIcal step Continuous rocking will prevent the cells from sticking to the bottom of the fask or dish. It is important to culture
cells for at least 2 h to recover some of the proteins that might have been affected
by the short-term trypsin treatment.
6. After incubation, count the number of cells and use them for selection, following the same procedure as described for suspension
cells (Steps 2–60).
5. For cells dissociated by short-term trypsin treatment, use cells immediately, as described (Steps 2–60), or incubate cells under cell
culture conditions, but with rocking for at least 2 h.
crItIcal step Continuous rocking will prevent the cells from sticking to the bottom of the fask or dish. It is important to culture
cells for at least 2 h to recover some of the proteins that might have been affected by the short-term trypsin treatment.
6. After incubation, count the number of cells and use them for selection, following the same procedure as described for suspension
cells (Steps 2–60).
crItIcal step Trypsin treatment must be performed with extreme caution because substantial damage to surface proteins can occur
if the treatment is prolonged. Moreover, do not treat cells with prolonged exposure to the harsh nonenzymatic dissociation buffer. Atscraper
the most, 5 min is suffcient. Both higher concentration and long-term exposure are detrimental to cell integrity and surface markers.
?  trouBlesHootInG
BoX 2 | PERFoRm SELECTIoN DIRECTLY IN CULTURE DISH ● tIMInG 1 H 40 mIN
1. Culture cells into monolayers with at least 90% confuence.
crItIcal step Performing selection directly in the culture dish maintains cells in their natural environment and proteins in their
native conformation. There is less concern of cell surface variability.
2. For the frst round of selection, use a 100-mm × 20-mm culture dish (cell number over 5 million). Wash cells with washing
buffer twice.
crItIcal step Do not use a culture fask when performing selection directly with adherent monolayer because it is easier to wash
and remove the cells from an uncovered dish than from a fask.
3. Prepare DNA library in 500 µl of binding buffer.
4. Incubate cells with DNA library. Use a total volume of 1,000 µl for the 100-mm ×20-mm culture dish and shake continuously
(50 ) or on slow rocker for 1 h.
crItIcal step Because the DNA library volume is expected to cover the entire culture dish, adjust the total volume of the DNA
library to 1,000 µl while maintaining the fnal concentration.
5. After incubation, wash cells three times with washing buffer.
crItIcal step Ensure that the cells are not detached during washing, as this will cause loss of binding sequences.
6. Add 500 µl of DNAse-free water to the washed cells. Detach cells using cell scraper and transfer them into a 1.5-ml tube.
7. Heat suspension at 95 °C for 10 min and recover the suspended DNA by centrifugation at 13,100g for 5 min. Ensure that the
recovery effciency is as high as possible.
8. Follow Steps 10–34 to obtain the frst selected DNA pool.
pause poInt One may store the DNA pool at  −  20 °C.
second round of selection
9. Use the generated frst-round selected pool, resuspended in binding buffer.
10. Follow Box 2, steps 1–5.
11. Elute the cell-DNA complex in 600 µl of binding buffer. Detach cells using cell scraper and transfer them into a 1.5-ml tube.
Heat and recover the eluted DNA suspension by centrifugation.
negative selection
12. Use a 100-mm × 20-mm culture dish with cells in confuence.
crItIcal step Avoid nonconfuent cell culture as DNA can stick to the bare culture dish.
13. Wash the cells.
14. Incubate cells with the eluted pool.
15. After incubation, recover the buffer containing the remaining sequences that do not bind to the control cells. Label this as the
second selected DNA pool.
pause poInt One may store the selected DNA pool at  −  20 °C.
pcr procedures—ssDna
16. Follow Steps 15–34 to obtain ssDNA
subsequent rounds of selection
17. Use a 60-mm × 15-mm culture dish to carry out the incubation step using 250 µl of the ssDNA pool with 250 µl of binding buffer
and maintaining a 1,000 nM fnal concentration of the selected DNA library. Follow with the washing and elution steps.
pause poInt One may store the selected DNA pool at  −  20 °C.
Monitor progress of selection
18. One may dissociate cells and use similar binding assays as described for suspension cells (Steps 52–59) or use confocal microscopy
when adherent.
1174 | VOL.5 NO.6 | 2010 | nature protocols
the purpose of selection. In general, any temperature between 4 and 37 °C is applicable for this protocol; however,
higher temperatures such as 37 °C can cause internalization. Once aptamer is selected at 4 °C, it is important to test
the binding at 37 °C. This does not mean that all aptamers selected at 4 °C will not bind at 37 °C. From observation, most
of the aptamers will bind very well at 37 °C, especially those with very high affnity. Some of the aptamers generated at
4 °C have been used in various applications at 37 °C (refs. 30,33). The most important point here is to generate aptamers
with high affnity by gradually increasing the stringency of selection to generate aptamers that can bind at diverse binding
conditions. For instance, aptamers that have been developed using DPBS as the binding buffer can still recognize the target
in culture medium.
crItIcal step Because of the large number of cells relative to the small volume of
incubation medium, it is sometimes
common to see cells settling at the bottom of the tube, even while shaking, so occasionally check and resuspend the cells.
Washing step ● tIMInG 40 min
7|  After incubation, centrifuge cells at 150g for 3 min at 4 °C. Remove supernatant containing unbound sequences and
resuspend cell pellets in 3 ml of washing buffer. Shake for about 30 s and centrifuge again using the same condition. Remove
supernatant carefully with transfer pipette, avoiding cell loss. Quickly spin down (30 s) the residual buffer on the wall of the
tube. To avoid cell loss, remove a small amount of residual buffer with folded kimwipe. Repeat the washing procedure two
more times for a total of three washings.
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elution of bound sequences ● tIMInG 15 min
8|  For the frst round of selection, elute the bound sequences in water. Add 500 µl of DNase-free water to the cell pellet.
Resuspend cells and transfer cell suspension into a 1.5-ml microfuge tube.
crItIcal step Only in the first round is DNA eluted in water. In subsequent rounds, DNA is eluted in binding buffer. As the
entire eluted pool is amplified by PCR, use only DNase-free water for the elution of the bound DNA sequences during the first
round of selection. Otherwise, if the DNA is eluted in DPBS, the salts with high concentration will affect the efficiency of PCR.
9| Heat the cell mixture at 95 °C for 10 min, centrifuge at 13,100g for 5 min and collect supernatant containing eluted DNA.
crItIcal step Do not perform negative selection at this point. Minimize the loss of eluted sequences during the first
selection round, as each sequence is theoretically represented only once, and when any sequence is lost, it can never be
recovered.
pause poInt Store eluted DNA at  −  20 °C.
pcr procedures: pcr amplification of the entire first selected pool ● tIMInG 1 h 10| This step is performed for pools generated from only the frst selection. Set up a 1,000-µl PCR amplifcation reaction
volume as shown below: adjust the volume of water to compensate for the total volume if the DNA selected pool
volume differs.
reagents reaction mixture (µl)
10× PCR buffer 100.0
dNTP mixture (2.5 mM each) 80.0
FITC- and biotin-primer mixture 50.0 (0.5 µM final)
DNA selected pool (template) 500.0
DNase-free water 270.0
HS Taq polymerase 3.0
11| Mix thoroughly and pipet 100 µl into 10 individual tubes (using 96-well thermal cycler) for 100 µl per reaction, or pipet
200 µl into fve individual tubes (60-well thermal cycler) for 200 µl per reaction.
crItIcal step Note that this step is only performed to step up the copies of individual sequences and is not necessar-
ily preparative. Too many cycles may produce nonspecific amplicons, and this will affect the purity of the DNA library pool.
Choose 8–10 cycles.
12| Perform PCR amplifcation. For example, in primer set 2 given above, use the following amplifcation conditions: denaturation
at 95 °C for 30 s, annealing at 56.3 °C for 30 s and elongation at 72 °C for 30 s, followed by fnal extension for 3 min at 72 °C.
crItIcal step Always keep Taq polymerase at  −  20 °C until ready to use. Thaw and keep all other PCR reagents on ice.
step temperature (°c) time (s)
Hot start 95 150
Amplification (10 cycles)
Denaturation 95 30
Annealing 56.3 30
Extension 72 30
Final extension 72 180
Hold  4
13| After PCR amplifcation, pool all reaction mixtures together (total 1,000 µl). There is no need to perform agarose gel
electrophoresis to assess template amplifcation. In most cases, the frst-round PCR amplifcation product is insuffcient for
detection by ethidium bromide.
crItIcal step Note that the PCR-amplified pool can potentially contaminate PCR reagents and primers; therefore, keep
them separated.
14| Label the PCR product as the frst selected pool.
pause poInt Store the DNA pool at  −  20 °C.
pcr procedures: determine the optimum number of cycles for preparative pcr ● tIMInG 1 h 30 min
15| The total PCR reaction mixture volume for each tube is 50 µl, with the amplifed frst selected pool at 10% serving as
the template. Choose PCR samples at the following cycles: 4, 6, 8, 10 and 12, and one negative control at the twelfth cycle.
Add the following reagents to PCR tubes as shown below:
reagents positive reaction mixture (µl) control (µl)
10× PCR buffer 25.0 5.0
dNTP mixture (2.5 mM each) 20.0 4.0
FITC- and biotin-primer mixture 12.5 (0.5 µM final) 2.5
Amplified DNA selected pool (template) 25.0 (10% of reaction mixture) —Supernatant of pure cell lysate — 5.0
DNase-free water 166.75 33.35
HS Taq polymerase 0.75 0.15
Abbreviation: FITC, fuorescein isothiocyanate.

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