13022013_LAC_B cell sorting

OBJECTIVE

To sort plasmablasts, naive B cells and memory B cells to see if we can detect antibodies against NF155 in these subsets.

MATERIALS

Same procedure as in the setting-up experiment

Only difference is that tubes came from Granada overnight and that they used Hep-Li tubes with plastic balls inside. That had to be removed before proceeding with Ficoll.

PROCEDURE

  •  Transfer all blood to 2 50mL falcons and spin down cells at 900g 10′
  • Remove plasma and store it frozen (Box with LAC plasma from Miquel)
  • Add RPMI (w/o additives) up to 50mL again and re-suspend.
  • Prepare 4 x50mL Falcons with 15mL Lymphoprep each.
  • Add 25mL of cell suspension to each Falcon w/ Ficoll and spin at 400g 20′ w/o brake
  • Get PBMCs and re-suspend in RPMI (the interphase of PBMCs is blurry. For some strange reason cells are not well separated). Spin down at 300g to wash (2x)
  • Re-suspend in 5mL and count (counted 100xmill cells total, aprox)
  • Prepare these tubes:

Tube 1: CD19-FITC only

Tube 2: CD27-PerCPCy5.5 only

Tube 3: CD38-PE only

Tube 4: Only cells

Tube 5: All antibodies

  • All compensation tubes 1mill cells (in 1mL)
  • The rest of cells (90mill aprox.) into 3mL of RPMI for sorting
  • Added 5uL of each antibody for compensation and 200uL for the sorting tube
  • Incubate Abs 30min in the dark
  • Wash 2x with RPMI
  • Prepare 3 tubes (CD19+CD27+CD38+, CD19+CD27+CD38-, CD19+CD27-CD38-) with basal medium
  • Go to sort
  • After sorting spin down cells at 400g and re-suspend in 100uL of basal medium plus IL6 (1ngr/ml) and count
  • Plate 100uL with all sorted cells from each subset in a 96-well plate with basal medium + IL6. Wait one week to collect supernatant.

RESULTS

  • Sample was extremely dirty. Many red cells, many dead cells.
  • Very few CD27+ cells. Concentrations of antibodies were the same as in the set-up experiment. Something went wrong with either the sample or the staining. It can be possible that the patient does not have many CD27+ B cells. We’ll know better after doing regular FACS tomorrow with the same sample.
  •  Plasmablast population was close to zero due to low CD19+CD27+  numbers.
  • The other two subsets had enough cells to culture (see reports below)

Global Sheet1_14022013143136 Sort_Report_14022013143055

CONCLUSIONS

We are not going to see anything in the plasmablast subpopulation…unfortunately.

Tomorrow I’ll do regular FACS to analyze B cell subsets and see the percentages of each subset to see if they match with the sorting.

Print Friendly, PDF & Email

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.