Honest protocols! Splitting Cells.

I'm starting a new theme on this blog: the honest protocols. 

These are accurate protocols on some of the things that scientists do on an almost daily basis - but with a twist. These honest protocols will describe the whirlwind of emotions that a scientist may experience along with the all the problems and delays that will inevitably happen even if the protocol sheet you were given doesn't tell you anything about them. I'm vaguely hoping it will be kind of educational for the budding scientists out there, maybe relatable for the experienced scientists and potentially insightful for those who are curious about what people might actually do in a laboratory. Hopefully, it will be a little funny!

First protocol up is Splitting cells. 

Splitting is what scientists have to go when their cells divide too much and need to be separated. Cells for experiments can be grown in these cell culture flasks that are designed to accommodate a certain number of cells based on the size. T75s can take about 8 million, and T25s can take about 3 million.  The cells are deposited in the flask along with some cell media that has all the nutrients they need, shoved in an incubator and left to it. The amount of cell media you can use is proportional to the size of the flask you use. So, if you let the cells divide for too long, you get too many in one flask and the media doesn't have enough nutrients to support them all.  This means that they will get stressed and start fighting. Sounds a bit like my old school's canteen at lunch hour. Cells being stressed can ruin a lot of data because they behave differently to unstressed cells. Explains most of my behaviour during my A levels to be honest. Anyway, if we want to keep our cells unstressed, we need to get them out of the flask and put some of them into a new flask with fresh media, where they can carry on dividing. Until we need to split them again. 

Cell culture flask sizes: The tiny one is T25 (surface area of 25cm^2), 

followed by a T75, a T175 and a T225. The lids often have a filter so the cells can get enough oxygen but stops anything that could contaminate the cells getting in. 

So step 1: Go to the incubator, locate your cells and check how they look under a microscope.  Cells usually stick to the inside of the flask and form a monolayer.  If you are going to split cells, you need to make sure you have enough of them to maintain a stable population- a small number of cells cannot always divide enough to give you enough cells for an experiment. But you also don't want to wait until the cells get stressed.  So, when looking at the cells, you want them to fill about 70% of your field of vision. 

This is about 70%. 'About'.

Step 2: Swear when you see that the cells haven't grown enough and you don't have enough for splitting.  You were going to use the left-over cells for a lysate and now because you can't split, you can't make the lysate- which you need for an experiment.

Step 3:  Experience the stages of grief and finally you accept that you can't split today. 

Step 4: Sulkily go to the freezer to see if maybe you froze some lysate you could use. You didn't.

Step 5: Stomp back to your desk to see if this delay is going to be completely mess up your life plan - or at least your plans for the next week. Grab a coffee on the way. Possibly a brownie if you need cheering up.

Step 6: Write up a new plan for the week and spend the rest of the day doing your data analysis. Who says you aren't flexible?

Step 7: It’s the next day! You slowly drag yourself up to the lab, bracing yourself for contamination and dead cells.  There's going to be a reason you can't do it today, you aren't going to be blessed with luck....

Step 8: Repeat step 1 - and the cells have grown! You're happy at first - and then sigh when you realise that you didn't book the cell culture hood in advance or warm up the reagents you will need. Cells can be fussy and don't like cold fluids poured on them. It can cause stress. Not sure I blame them.

Step 9: By some miracle, the cell culture hood booking sheet is empty*

*The cell culture hood is one of the most important pieces of equipment for anyone working with cells. It creates a sterile (clean) environment meaning you can open up your cell culture flask and know that unless you screw up, no viruses, bacteria or fungi can get into your cells and mess up your work. 

Standard cell culture hood. 

 You book yourself in for a slot that starts in about half an hour and start getting your reagents out of the fridge. You need Trypsin; an enzyme that is designed to get the cells of the walls of the flask.  Enzymes are made out of protein which get a bit deformed if too hot so that one can sit out at room temperature.  You need PBS - we use this to wash the cells from any proteins that interferes with the trypsin and keep the cells hydrated. You also need your cell media, which has all the components for healthy cell growth. You can add something called FBS which provides some extra proteins and some extra help - like taking multivitamins alongside a healthy diet. Antibiotics are also added to kill any bacteria.

Step 10: Wipe the entire hood down with blue roll* and ethanol. This is to kill anything that might be in the hood. The hoods do have UV light - which also kills everything and filter air, but you don't know if the person who used the hood before you actually used the hood properly. 

*What no one tells you before you step into the lab is that blue roll is actually the most important thing in a lab. Doesn't matter how many cells you have or what equipment you have, if you don't have blue roll, you are SCREWED. 

It's the exact same stuff that bars and kitchen use. 

Step 11: Remove the cell media from the cells.  For this you will be using a pipette buoy and a stripette. 

Pipette buoy on the left and a stripette. The stripette attaches to the buoy 

and the two buttons on the buoy control whether fluid is sucked up the stripette

 or down the stripette 

               
                                    

Step 12:  Wash the cells with PBS. If using a flask, you want anything from 5-10 ml depending on size of flask.  Question whether you sprayed your gloves down with ethanol before opening the flask.

Step 13: Pipette Trypsin onto the cells. 1-2 ml is usually fine. 

Step 14: Incubate for 3 minutes in the incubator.  Use this time to dead-scroll Facebook, mull over what you're having your lunch, question your life choices or make notes in your lab book. Realise that you didn't take your gloves off before touching your phone and you will have to put new gloves on before touching the flasks again. 

                                                        

Step 15. Check for a thick white fluid - this is the cells coming loose. Bang the flask on a table to shake them all loose- wince when you realise you should have 'tapped' and not 'banged'. Head back to the hood. Almost forget to spray the flask with ethanol. 

Detaching: When cells come loose. 

Can also tap the flask with your hand.

Step 16. Using your trusty pipette buoy, add your fresh cell media to the flask.  About 8mls. Transfer it all to a 15 ml tube. 

Step 17: Put 15 ml tube in centrifuge* Realise you need a second tube to balance it out. 

*Centrifuges are used to separate based on density. So, in this case, you want to get all the cells separated from the media and the trypsin. Centrifuges use gravity and centripetal force to pull the densest components down first, and because the cells are the heaviest part in this mixture, they will form a white pellet at the bottom of the tube. But because they use centripetal force, you need the centrifuge to be balanced either side. Which means we need another tube of the same weight. Shift though all the blanks* that some kind, thoughtful and organised lab technician made up.

*Blanks are a general term basically describing something that won't contribute to your results or give you data but will get your equipment working. In this case, the blank is a 15 ml tube containing some water. If we have 10ml of cell media + cells in a tube, we need a blank with 10ml of water. 

Step 18: After questioning your sanity for about 5 minutes, you find the blank. Shut your centrifuge, set it 1200 rpm at 4 minutes. Pray to whatever higher power you choose that it's balanced properly and today you won't hear the god-awful grating grinding sound of an unhappy centrifuge that makes you regret all of your choices. Use this time to mentally thank lab techs and return to dead scrolling.

*rpm means rotations per minute. Just tells you how fast the centrifuge is going. Honestly, the cells aren't that fussy, and the rpm doesn't actually matter that much. Just keep it below 1500 to stop them being damage.

Step 19: Remove the tube, check that you have a pellet. You do. If you haven't, something MAJORLY WRONG has happened. 

Step 20: Return tube to cell hood. Use pipette to remove the liquid - leaving the pellet behind. Accidently poke pellet with pipette edge. Tell yourself that its fine *.

*It 'probably' is.

Step 21: Curse when you realise that you didn't check whether the flasks you needed were actually in the flask storage drawer. They aren't.

Step 22: Run across to the storage room and grab a new bag. 

Step 23: Label your flask with your initials, the data, the type of cells and their passage number*.

*Passage number is how many times the cells have been split. If their passage number gets too high, they can get mutated and behave weirdly. 

Step 24: Pause as you try to remember what's the date and the passage number. Ask kind lab tech who walks in.  

Step 25: Consider if you have cool initials or whether your initials are just weird*

*I knew someone whose initials were EW. Her flasks were literally EW. 

Step 26: Pipette media into the new flask - about 9ml. Idly consider how when the lab tech showed you how to do this, it took them 5 minutes to do the whole thing and it has currently taken you about 15 minutes to even get to this point. 

Step 27: Return to pellet and resuspend it with 10 ml of media.

Step 28: Transfer 1 ml of this to the new flask, and add 9ml of fresh media*

*This means that you have one tenth of the cells growing in the old flask now growing in the new flask. 

Step 29: Make your lysate- with the remaining cells*.

*Honest protocol 2. 

Step 30: You decide to make up some media in advance, so hunt down your FBS from the fridge.

Step 31: You swear when you realise you used up your FBS last week.

Step 32: Retrieve the 50ml tube FBS from the freezer and mutter angrily when you realise that you need an hour to defrost it. 

Step 33: Scrawl your name on another slot on the booking sheet in an hour's time. Stuff the FBS in the water bath to defrost. Wipe everything down with ethanol.

Step 34: Come back to the lab after knocking back a coffee. Wonder if your entire blood stream is just pure coffee and sugar. 

Step 35: Wipe everything down with ethanol. Consider the sad fact that you have had to wait an hour to do something that will take you less than 5 minutes and it’s your poor planning and lack of forward thinking that led you to this. 

Step 36:  Pour the FBS into the media*

*You could actually be a bit more accurate if you feel like it. The media usually comes as 500ml and you need about 10% of FBS.  About. So, you could pipette 50ml of media out and replace it with the FBS or just shove in 50ml of FBS. 

Step 38: Return the cell media to the fridge. 

Step 39: Almost forget to put an extra FBS in the fridge- ALMOST. Grab the frozen FBS, shove it in the fridge.  You can use it next week when you need to make more media. For the tenth time this week, silently wonder how you manage to use up so much media. 

Step 40: Knock back another coffee... sigh and start planning the experiments you are going to do...