做cell biology实验，细胞铺板大概是最常见的一个实验了。但是有很多人不是很得要领，铺得不是均匀： 要么中间密周围稀，要么周围密中间秃顶。
你有哪些看家技巧？不妨拿出来和大家分享一下。有奖励。



以前分享的一些技巧：
1、一般96孔板我每孔是加100微升细胞悬液，从孔的左边靠近底部加入，加完半边板后，将未加的细胞悬液混一下再，继续加剩余的半边板子，都加完后盖上盖子，左手轻轻扶住板的左边，右手轻轻敲击板的右边缘，注意把握力度（我一般轻巧敲三下），太强或次数太多会导致细胞集中成堆，将板顺时针旋转（逆时针效果不好），依次敲击剩余三个边，静置约5分钟，放入37度培养箱。

6孔板12孔板或24孔板，我均采用将第一个孔加入少量无血清培养基，晃动浸润整个孔底，然后用移液*吸至第二孔，同样方法浸润孔底，其它孔一次类推，这样整个孔底都是湿润的，细胞悬液会平铺在整个孔底，加细胞悬液的时候可以避免加在中间中间细胞多，而加在周边晃匀后周边细胞多中间少的现象，细胞分散较均匀，注意加完细胞悬液后要放工作台静置一下。这个方法就是有点慢，但操作熟练了也不慢。也可以采用轻拍的方式，但力度没有96孔板好掌握，效果没有96孔板好，所以我放弃改用浸润孔底的方法。

2、细胞悬液加完后，将细胞培养板抬高，对着灯光，从底部往上看，看细胞有没有抱团。然后从底部敲击，使之分散。

3、如果实验室有平板振荡器的话，我建议用这个仪器稍振荡一下，效果不错，就是振幅小，频率高的那种。

4、细胞要尽量打散，大部分呈单个状态。离心后，要充分悬浮！还有转移到六孔板后，是要晃得！晃的时候最好不要让那个细胞液转圈，不然细胞就全被带到中间去了，就会不均匀！

5、一瓶细胞长满后，正常处理，在培养瓶里吹匀，然后铺6孔板，每孔2毫升，铺完之后不用观察直接用酒精棉擦拭，然后放到培养箱里，轻微的左三圈 右三圈 前三圈 后三圈。基本上24小时之后观察 每孔的细胞都会很均匀。

6、计算好所需要的全部液体量和细胞量，混匀后，加到六孔板里，六孔板按横8字型晃，显微镜下观察，如果不均匀，按上述方法再晃。如果细胞未计数直接种的话，在种六孔板的过程中，随时晃一下混匀用的瓶子，瓶子我通常是顺时针或逆时针转圈。

7、放在水平板面上先上下移动，再左右移动，每个方向５到６次，但关键的是摇完后最好直接放入培养箱中，不要再做过多的运动，例如放到镜下去看，否则很容易就又聚到中间去了。

8、老外的技巧：
＃1: Our lab found an even plating to be accomplished by moving plates backward and forward, then right to left to right, repeat same motions x 5. Next, allow plates to sit for 3-5 minutes before placing into incubator. We found that any type of swirling would cause the cells to either congregate in the center or along the outside of the wells.

＃2: never swirl to mix multi well plates. I always slide the plate back and forth, left and right about 5 - 10 times in each direction, taking care not to introduce any circular motion into the media. Swirling causes the media to move in a circular motion, and much like sand in a cup of swirling water, it moves the particles to the center. The idea is to limit any type of circular motion. t25 flasks etc with edges don't have this problem, because circular motion of the media is harder to propagate and maintain. Sometimes multi-well plates with square wells work well for this as well. Nunc makes some multiwell TC plates with rectangular wells.

＃3: The outermost wells in a plate are more prone to frequent changes in conditions such as temperature. You should try preincubating the plate along with medium for 10-15 minutes before plating your cells.

＃4: When you rotate plates horizontally, cells tend to gather at the center of well. It is suggested shaking a plate back and forth and right-and-left. If you use less medium, more medium stay close to the wall of a well. This could help to avoid that cells to go to the center of a well.
I usually try less medium to seed cells, I rotate plates horizontally first, and then incubate it for 1 min in an incubator. As a result, cells gather at the center of well, but I shake it again in a back-and-forth-and-right-and-left manner this time. The reason I incubate it for 1 min is that cells get down to the surface of the bottom. This means that cells are ready to attach the surface soon. When you distribute cells evenly under this condition, cells attach before circulation of media bring cells to specific locations.

＃5: It is a common issue due to vibration in incubators. Easiest method to overcome this issue - after adding the cell suspension to wells (minimal volume till cells get attached) gently swirl the plate to spread the cells evenly. Keep the plate on a flat surface for 5 minutes (bench top in the tissue culture room) . Then transfer to an incubator. Also make sure the trays in the incubator are horizontal. Keeping more flasks on one side may tilt the tray towards the heavier side.

＃6: When you plate your cells make movements in a figure of eight, I mean, as if you were picturing an eight...ten times. With this movement I resolved my problem...

＃7: Mix cell suspension, add to the wells of 24 well plate and leave the plate at horizontal surface at room temperature for 1 hour prior to placing the plate to CO2 incubator. Following this technique should greatly improve even cells distribution across the wells.

＃8: Laminar flow hood benches produce vibrations that cause cells to settle unevenly. You don't want the cells cells settling in there if you can avoid it. Incubators also have vibrations. So putting cells too soon into the incubator can be the issue. You may even be encountering convection currents in the media as the temperature equilibrates to the incubator which will move unattached cells around. This may explain the different cell patterns on the edge and center wells of the 24-well plate. These two problems can be solved by settling and attaching cells on the bench for 20 min.
Another possibility is you have poorly coated tissue culture plates. In that case change your supplier. Corning is reliable for having good coatings on tissue culture plates.
Also keep your volume around 0.5ml in 24-well plates. Minimal volumes will give you a ring of denser cells around the edge due the meniscus effect.

＃9: .in a simpler manner.....you can 1st add slight medium in the well without cells.....it should cover the surface of well......and after covering the surface of well with medium ....now add your cell suspension into the medium and mix with pipette gently thrice.....it will not attach mid of the well and distribute all over the surface.

＃10:  just shake the plate every 10-15 min for the first 60-90 min.
That'll improve your outcome

＃11:  Doing only figures of 8 did improve the outcome but cells in some of the wells (usually the first few that were seeded) would still group in the center. What worked nicely for me; Holding both the 24 well plate and the tube of suspension in one hand, leaving the other hand free to pipette. Slowly tilting the plate back and forth continuously whilst the cells were seeded to keep them in suspension until I'd finnished. After the 24 wells are filled I leave them on the workbench outside of the hood (to avoid the vibrations) and do several gentle firgures of 8 and leave for 5-10 minutes, then check under the microscope before putting them into the incubator. The cells seemed eager to attach and not setting the plate down under the hood when seeding seemed to make a difference.
Also, putting a piece of tissue down underneath the plate when doing the figure of 8 motions not only avoided that aweful scraping noise but made the movements a lot smoother and so helped to avoid that edge effect that the figure of 8 can give you sometimes.

＃12: I always slide the plate back and forth, left and right about 5 - 10 times in each direction, taking care not to introduce any circular motion into the media. Swirling causes the media to move in a circular motion, and much like sand in a cup of swirling water, it moves the particles to the center. The idea is to limit any type of circular motion. t25 flasks etc with edges don't have this problem, because circular motion of the media is harder to propagate and maintain. Sometimes multi-well plates with square wells work well for this as well. Nunc makes some multiwell TC plates with rectangular wells.

9、your tips ?