In vivo B-cell Activation

[TheCroatian]

Hi!

 

I'm new to these forums and thought I would try my luck with a question.

I'm a student trying to design an experiment.

 

I have a large complex that I suspect induces a T-independent immune response. I want to induce B-cells in vitro that respond to this antigen. How would I go about setting up this experiment?

 

Thanks so very much to the kind person who can help me out

Edited July 8, 2008 by TheCroatian

[jtendres]

Hi there. I hope this helps. I did an experiment in an immunology class that involved stimulating B-cells with pokeweed mitogen and lipopolysaccharide and then to see if proliferation occured, we did a conjugate-enzyme type reaction. Here are the instructions for the experiment for to figure out what is helpful.

 

PLATING CELLS

 

 It is critical that great care be taken to prevent contamination of the cultures. Precautions include:

 Place poly-backed absorbent lab paper under your work area

 Wear gloves that have been wiped with alcohol soaked gauze

 Wear a mask

 Wipe micropipette with alcohol soaked gauze before inserting into cell suspensions or treatment vials.

 Close pipette tip box immediately after retrieval of each tip

 Close lids on pipette tip boxes immediately after use.

 Do not set the lids of the TC plates down; hold while pipetting and then replace the lid between while retrieving the next aliquot.

 

1. When your counts are complete and calculations reviewed by an instructor, you will receive a 1ml aliquot of PBMLs.

 

2. After your cells arrive, but before you begin working with the cells, have everything ready at your desk

 

a. Wear gloves cleaned with alcohol gauze

b. Wear a mask

c. Open you pack of sterile, absorbent lab paper

 

3. Loosen the lids of both the Media and the “plating” aliquot (labeled PBML – Equine A or B) lightly (not so much as to risk the lid falling off!)

 

4. Using a 1 or 2ml sterile pipette and aseptic technique, remove the appropriate volume of DMEM+FBS from the “Media” tube. DO NOT SET THE CAP OF THE MEDIA TUBE DOWN! Remove the previously determined media volume (see step #5 in “cell counting”). and immediately replace the tube cap. Then remove the cap from the plating tube and add the media to it. Again, DO NOT SET THE CAP DOWN! The “plating” tube should now contain cells in a final concentration of 2e6 / ml.

 

5. Remove T.C. Plate from its package and set onto your lab paper.

 

6. Ensuring the tube lid is secure, gently vortex the tube to resuspend the cells then place the tube back into your rack. You must be ready to immediately proceed to the next step, as cells will settle fast.

 

7. Confirm that your micropipette is set to a volume of 100μl. Use an ETOH soaked gauze pad and wipe the shaft of the pipette (the end that holds the tip).

 

8. Loosen the lid of your PBML “plating” tube (not so much as to risk the lid falling off!). Then lift the lid of the sterile, plugged pipette tips, remove a tip. Immediately close the lid of the pipette box (you will share a box with your partner).

 

9. Remove the lid from the PBML tube and remove 100µl of cells. DO NOT SET THE CAP DOWN and IMMEDIATELY RECAP THE TUBE!

 

10. Lift the lid of the plate only enough to add cell suspension, pipette 100µl of cells (100μl of a 2e6 cells suspenstion = 2e5 cells / well) into 3 sets of wells (3 wells / set for a total of 9 wells) and IMMEDIATELY REPLACE THE PLATE LID; DO NOT SET THE LID DOWN. A suggested distribution pattern will be suggested the day of class.

 

11. Using aseptic technique, add 100ul of each treatment (3 wells receive TC media, 3 wells receive ConA and 3 wells receive LPS) to appropriate wells. Treatments (ConA & LPS) are made up at twice the desired final concentration as the volume is diluted 1:2 by the 100ul of cells already in the wells. The snap cap tube lids are difficult to place on and off with 1 hand, therefore you may set the cap down, however you must immediately pipette the treatment into all 3 well, following the same protocol for adding cells to the plate (Step #10). Again DO NOT SET PLATE LID DOWN!!

 

12. Once complete, give the plate to an instructor so they can incubate them for 72hrs at 37ºC and 5% CO2 before pulsing with 20µl BrdU (dil 1:100 as described in kit). The cells will then be incubated an additional 18hr and then spun at 300xg (1400rpm). Supernatant is removed and plates dried (blown with a hair dryer for 15min). The plates will be stored at 4°C (refrigerator) until the next laboratory session.

 

1. Add 200μl of Fix/Denaturation solution into each appropriate well (previously containing cell culture) and incubate for 30minutes at RT (at your bench).

 

2. Remove Fix/Denaturation solution by flicking off excess into sink and then tapping plates firmly, but gently onto paper towels until most of the liquid has run out of the plate (do not let wells dry).

 

3. Add 100μl of anti-BrdU-POD solution and incubate at RT for 1hr.

 

4. Remove antibody conjugate by flicking off excess solution into the sink and then tapping plate onto a paper towel as in step #2.

 

5. Add 200μl of Wash Buffer and let sit for 5 minutes. Shake off excess Wash Buffer into the sink and tap plate onto paper towel to remove excess.

 

6. Repeat the Wash step twice more (Step #5; total of 3 washes).

 

7. Add 100μl of Substrate Solution (see instructor). Incubate for 15 minutes.

 

8. Add 25μl of Stop Solution, mixing each well as added and changing tips between each well.

 

9. Read immediately on ELISA-reader using a 450 nm filter. The reference wavelength should be 650 nm. The computer will automatically subtract the readings of the reference wavelength from that of the 450nm filter. An instructor will have the machine set up to read in this fashion and will provide a computer printout of your results.

 

10. After obtaining the printout of your O.D., calculate the average OD for each treatment (add the 3 together and divide by 3). If any one of the readings is extremely different form the other 2 (>2X) then discard it, however if all 3 readings are vastly different you have no choice but to average all 3.

 

11. Determine the Stimulation Index (SI) for each treatment.

 

a. To determine the SI, divide the treatment OD (avg) by the average media control OD.