CEC Assay

Author

Brian

Published

February 22, 2023

The current optimized CEC assay is a two-day experiment.

Cell Plating (Day before experiment ~ 4 pm)

  1. Plate Microglia/J774A1 cells at 50,000 cells per well in 96-well black-clear bottom plate in complete media

    1. Complete Media = DMEM or RPMI, Fetal bovine serum (10%), Streptomycin + penicillin (1%)

  2. NOTE: Outer well should be blocked

Example of plate layout on 96-well

  1. Incubate for 18 Hours

    1. REASONG BEHIND Long Incubation

      1. 18 hours is the doubling time for J7741A cells so final concentration is ~100,000 cells per well next day.

      2. Cells are more adherent with overnight plating, more robust, CVs very nice.

Cholesterol loading (Day of experiment ~9 am)

Reagent Mixture Preparation

  1. Prepare cholesterol loading mix (100 ul/well) with the following:

    1. BODIPY-cholesterol (Kit): 1:4

    2. Equilibrium Media (Kit): 1:4

    3. SERUM FREE RPMI MEDIA (No FBS/antibiotics): 2:4

    4. Reagent A (ACAT Inhibitor or cAMP): 1 ul per 1mL of Serum Free Media used

    5. Reagent B (ACAT Inhibitor or cAMP): 1 ul per 1mL of Serum Free Media used

Reagent Recipe

Code 
library(tidyverse)
library(DT)
df <- tibble(sample = 1:96) |>
        mutate(sample_10_per = sample * 1.10,
               total_vol = sample_10_per * 100,
               SFM = total_vol/2,
               BODIPY = total_vol/4,
               Equ_media = total_vol/4,
               reagentA = SFM/1000,
               reagentB = SFM/1000) 

datatable(df, colnames = c("# Sample", "10% Extra","Total Vol. (uL)", "Serum Free Media (uL)"," Bodipy Cholesterol (uL)","Equ. Media (uL)"," Reagent A (uL)","Reagent B (uL)"))

Cholesterol loading

  1. Wash cells with SERUM FREE RPMI MEDIA x 1, flick it in the sink

  2. Load cells with 100 uL of cholesterol loading mix

  3. Incubate for 4 Hours

Prepare Acceptor at least 1.5-2HR before the end of loading

HDL as Acceptor

  1. HDL protein concentration should be measured by microBCA ahead of time

    1. 10 ug of HDL or 100 ug/mL HDL per replicate

    2. Total volume should be 115 uL per well

      1. REASON BEHIND THIS

      2. CVs are more consistent when you have more volume inside well, evaporation has minimal effect.

115 uL of total volume/replicate with FLUOROBRITE DMEM (PHENOL RED FREE) SERUM FREE MEDIA

  1. REASON BEHIND THIS (FLUOROBRITE DMEM)

    1. This special media has minimal background fluorescence.

  2. E.g. Subject 1 has 3000 ug/mL of HDL. You have one 25 uL aliquot to use for this assay. The formula goes like this for duplicates (and a little extra)

    1. Total Volume per well: 2.25 replicates x 115 uL total volume = 258.75 uL

    2. 3000 ug/mL * x =  258.75 uL * 100 ug/mL

    3. x = 8.62 uL of isolated HDL

    1.  258.75 - 8.62 uL HDL = 250.13 uL FluoroBrite media

    1. This means for this subject, mixed in a 1.5 mL tube:

      1. 250.13 uL of FluoroBrite Media

      2. 8.62 uL of isolated HDL

ApoB-depleted Plasma As acceptor

  1. Prepare 2% ApoB-depleted plasma in 115 uL of FluoroBrite media

    1. E.g. 2% of 115 uL = 2.3 uL of ApoB-depleted plasma per replicate

    2. 2.25 replicate x 115 uL total volume = 258.75 uL

    3. 2.3 uL ApoB-depleted plasma * 2.25 = 5.2 uL ApoB-depleted plasma

    4. Mix together in 1.5 mL Tube

      1. 5.2 uL Apob-depleted plasma

      2. 253.6 uL FluoroBrite media

Acceptor loading

  1. Wash cells with FLUOROBRITE DMEM (PHENOL RED FREE) SERUM FREE MEDIA x1.

    1. FBS has lipoproteins, so you don’t want your efflux to be confounded by the FBS

  2. Load cells (115 uL of HDL/FluoroBrite media mixture) per well.

  3. Cover with Breath-Easy Film

    1. REASON BEHIND THIS (Breath-Easy Film)
    1.  Minimizes evaporation

  4. Incubate 4 Hours

 

Remove Supernatant and Lyse Plate

  1. Remove supernatant into 96-well black-opaque bottom plate (note black-opaque bottom and not clear bottom

  2. Lyse remaining cells with 115 uL of M-PER lysis reagent

  3. Put lyse fraction in shaker for 30min no temperature

    1. No need to transfer this lyse fraction, keep in original plate

Reading - Supernatant Fraction

  1. Read supernatant in Microplate reader Ex/Em = 485/523 nm

  2. Settings for Supernatant fraction

    1. Set Plate to Black-opaque bottom

    2. 3 Reads fluorescence Endpoint

Reading - Lyse Fraction

  1. Read Lyse Fraction in Microplate reader Ex/Em = 485/523 nm

  2. Settings

    1. Set Plate to Black-clear bottom

    2. 1 Read on fluorescence AREA SCAN

    3. Extended Dynamic Range

    4. OPTIONAL:

      1. 3 additional reads on fluorescence Endpoint